Thermophilic and thermoacidophilic metabolism genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

ABSTRACT

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from  Alicyclobacillus acidocaldarius  are provided. Further provided are methods for modulating or altering metabolism in a cell using isolated and/or purified polypeptides and nucleic acid sequences from  Alicyclobacillus acidocaldarius.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/347,903, filed Nov. 10, 2016, pending, which is a continuation ofU.S. patent application Ser. No. 14/887,073, filed Oct. 19, 2015, nowabandoned, which is a divisional of U.S. patent application Ser. No.14/226,573, filed Mar. 26, 2014, now U.S. Pat. No. 9,222,094, issuedDec. 29, 2015, which is a divisional of U.S. patent application Ser. No.12/380,551, filed Feb. 26, 2009, now U.S. Pat. No. 8,728,803, issued May20, 2014, which application claims the benefit of the filing date ofU.S. Provisional Patent Application Ser. No. 61/032,339, filed Feb. 28,2008, for “THERMOPHILIC AND THERMOACIDOPHILIC METABOLISM GENES ANDENZYMES FROM ALICYCLOBACILLUS ACIDOCALDARIUS AND RELATED ORGANISMS,METHODS,” the disclosure of each of which is hereby incorporated hereinin its entirety by this reference.

GOVERNMENT RIGHTS

This invention was made with government support under Contract NumberDE-AC07-99ID13727 and Contract Number DE-AC07-051D14517 awarded by theUnited States Department of Energy. The government has certain rights inthe invention.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS TXT

Pursuant to 37 C.F.R. § 1.821(c) or (e), a file containing a TXT versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates generally to biotechnology. Morespecifically, embodiments of the present invention relate to isolatedand/or purified polypeptides and nucleic acid sequences encodingpolypeptides from Alicyclobacillus acidocaldarius and methods for theiruse.

BACKGROUND

Enzymes have a great deal of potential for production of usefulchemicals in industrial processes. However, industrial processestypically occur at extremes of temperature, pH, salt, etc., to whichmost of the well-studied enzymes and organisms are not well suited.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention relate to purified and/or isolatednucleotide sequences of the genome of Alicyclobacillus acidocaldarius,or a homologue or fragment thereof. In one embodiment of the invention,the nucleotide sequence is selected from at least one of SEQ ID NOS:2,19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257,274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495,512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733,750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954, 971,988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158, 1175,1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362, 1379,1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and 1566 ora homologue or fragment thereof. In another embodiment of the invention,the homologue is selected from the group consisting of a nucleotidesequence having at least 80% sequence identity to at least one of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954,971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158,1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362,1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and1566.

Embodiments of the invention may further relate to an isolated and/orpurified nucleic acid sequence comprising a nucleic acid sequenceencoding a polypeptide selected from the group consisting of apolypeptide having at least 90% sequence identity to at least one of SEQID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222,239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460,477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698,715, 732, 749, 766, 783, 800, 817, 834, 851, 868, 885, 902, 819, 936,953, 970, 987, 1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123, 1140,1157, 1174, 1191, 1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327, 1344,1361, 1378, 1395, 1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531, 1548,and 1565.

Embodiments of the invention also relate to isolated and/or purifiedpolypeptides coded for by a nucleotide sequence comprising a nucleotidesequence of the genome of Alicyclobacillus acidocaldarius, or ahomologue or fragment thereof. In one embodiment, the nucleotidesequence comprises a nucleotide sequence selected from the groupconsisting of a nucleotide sequence having at least 80% sequenceidentity to at least one of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121,138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359,376, 393, 410, 427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597,614, 631, 648, 665, 682, 699, 716, 733, 750, 767, 784, 801, 818, 835,852, 869, 886, 903, 920, 937, 954, 971, 988, 1005, 1022, 1039, 1056,1073, 1090, 1107, 1124, 1141, 1158, 1175, 1192, 1209, 1226, 1243, 1260,1277, 1294, 1311, 1328, 1345, 1362, 1379, 1396, 1413, 1430, 1447, 1464,1481, 1498, 1515, 1532, 1549, and 1566.

In another embodiment of the invention, the nucleotide sequencecomprises a nucleotide sequence selected from at least one of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954,971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158,1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362,1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and1566 or a homologue or fragment thereof. In still another embodiment,the polypeptide comprises an amino acid sequence of SEQ ID NOS:1, 18,35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273,290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511,528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749,766, 783, 800, 817, 834, 851, 868, 885, 902, 819, 936, 953, 970, 987,1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123, 1140, 1157, 1174, 1191,1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327, 1344, 1361, 1378, 1395,1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531, 1548, and 1565. In yetanother embodiment, the polypeptide comprises an amino acid sequenceselected from the group consisting of a polypeptide having at least 90%sequence identity to at least one of SEQ ID NOS:1, 18, 35, 52, 69, 86,103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 324,341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545, 562,579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, 766, 783, 800,817, 834, 851, 868, 885, 902, 819, 936, 953, 970, 987, 1004, 1021, 1038,1055, 1072, 1089, 1106, 1123, 1140, 1157, 1174, 1191, 1208, 1225, 1242,1259, 1276, 1293, 1310, 1327, 1344, 1361, 1378, 1395, 1412, 1429, 1446,1463, 1480, 1497, 1514, 1531, 1548, and 1565.

In embodiments of the invention, the polypeptides may be acidophilicand/or thermophilic. In further embodiments, the polypeptides may beglycosylated, pegylated, and/or otherwise post-translationally modified.

Embodiments of methods include methods of altering metabolism in a cell,the methods comprising providing a recombinant, purified, and/orisolated nucleotide sequence comprising a nucleotide sequence selectedfrom the group consisting of a nucleotide sequence having at least 90%sequence identity to at least one of the sequences of SEQ ID NOS:2, 19,36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274,291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495, 512,529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733, 750,767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954, 971, 988,1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158, 1175, 1192,1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362, 1379, 1396,1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and 1566 and/or arecombinant, purified, and/or isolated polypeptide selected from thegroup consisting of a polypeptide having at least 90% sequence identityto at least one of the sequences of SEQ ID NOS:1, 18, 35, 52, 69, 86,103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 324,341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545, 562,579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, 766, 783, 800,817, 834, 851, 868, 885, 902, 819, 936, 953, 970, 987, 1004, 1021, 1038,1055, 1072, 1089, 1106, 1123, 1140, 1157, 1174, 1191, 1208, 1225, 1242,1259, 1276, 1293, 1310, 1327, 1344, 1361, 1378, 1395, 1412, 1429, 1446,1463, 1480, 1497, 1514, 1531, 1548, and 1565 to the cell.

Further embodiments of methods include placing a cell producing orencoding a recombinant, purified, and/or isolated nucleotide sequencecomprising a nucleotide sequence selected from the group consisting of anucleotide sequence having at least 90% sequence identity to at leastone of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138,155, 172, 189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376,393, 410, 427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597, 614,631, 648, 665, 682, 699, 716, 733, 750, 767, 784, 801, 818, 835, 852,869, 886, 903, 920, 937, 954, 971, 988, 1005, 1022, 1039, 1056, 1073,1090, 1107, 1124, 1141, 1158, 1175, 1192, 1209, 1226, 1243, 1260, 1277,1294, 1311, 1328, 1345, 1362, 1379, 1396, 1413, 1430, 1447, 1464, 1481,1498, 1515, 1532, 1549, and 1566 and/or a recombinant, purified, and/orisolated polypeptide selected from the group consisting of a polypeptidehaving at least 90% sequence identity to at least one of the sequencesof SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205,222, 239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443,460, 477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681,698, 715, 732, 749, 766, 783, 800, 817, 834, 851, 868, 885, 902, 819,936, 953, 970, 987, 1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123,1140, 1157, 1174, 1191, 1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327,1344, 1361, 1378, 1395, 1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531,1548, and 1565 in an environment comprising temperatures at or aboveabout 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95degrees Celsius and/or a pH at, below, and/or above 8, 7, 6, 5, 4, 3, 2,1, and/or 0.

These and other aspects of the invention will become apparent to theskilled artisan in view of the teachings contained herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B depict a sequence alignment between SEQ ID NO:1(RAAC00079) and ref|YP_074710.1|, ref|YP_359514.1|, ref|YP_516748.1|,ref|YP_643635.1|, and ref|YP_144514.1| (SEQ ID NOS:3-7, respectively),which all have the function assigned to SEQ ID NO:1 in Table 1. Aminoacids conserved among all sequences are indicted by a “*” and generallyconserved amino acids are indicated by a “:”.

FIG. 2 depicts a sequence alignment between SEQ ID NO:18 (RAAC00455) andgb|ABE97159.1|, ref|NP_693902.1|, ref|YP_521150.1|, ref|ZP_01725542.1|,and ref|ZP_01666741.1| (SEQ ID NOS:20-24, respectively), which all havethe function assigned to SEQ ID NO:18 in Table 1. Amino acids conservedamong all sequences are indicted by a “*” and generally conserved aminoacids are indicated by a “:”.

FIGS. 3A and 3B depict a sequence alignment between SEQ ID NO:35(RAAC00461) and ref|YP_361350.1|, ref|NP_244632.1|, ref|ZP_00538452.1|,ref|YP_001127398.1|, and ref|YP_149222.1| (SEQ ID NOS:37-41,respectively), which all have the function assigned to SEQ ID NO:35 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 4 depicts a sequence alignment between SEQ ID NO:52 (RAAC00481) andref|NP_905294.1|, ref|ZP_01666099.1|, ref|YP_360429.1|,ref|YP_754604.1|, and ref|YP_384529.1| (SEQ ID NOS:54-58, respectively),which all have the function assigned to SEQ ID NO:52 in Table 1. Aminoacids conserved among all sequences are indicted by a “*” and generallyconserved amino acids are indicated by a “:”.

FIG. 5 depicts a sequence alignment between SEQ ID NO:69 (RAAC00529) andref|YP_146903.1|, ref|YP_001125035.1|, ref|YP_001646604.1|,ref|YP_001375911.1|, and ref|ZP_01696300.1| (SEQ ID NOS:71-75,respectively), which all have the function assigned to SEQ ID NO:69 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 6 depicts a sequence alignment between SEQ ID NO:86 (RAAC00552) andref|YP 001376041.1|, dbj|BAB39458.1|, ref|NP_846569.1|,ref|YP_896466.1|, and ref|ZP_00238879.1| (SEQ ID NOS:88-92,respectively), which all have the function assigned to SEQ ID NO:86 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 7 depicts a sequence alignment between SEQ ID NO:103 (RAAC00553)and ref|YP_001646745.1|, ref|YP_001376045.1|, ref|NP_833836.1|,ref|ZP_00739346.1|, and ref|YP_085454.1| (SEQ ID NOS:105-109,respectively), which all have the function assigned to SEQ ID NO:103 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 8 depicts a sequence alignment between SEQ ID NO:120 (RAAC00554)and ref|YP_147981.1|, ref|NP_390900.1|, ref|ZP_01667656.1|,sp|P228061BIOF_BACSH, and dbj|BAB39457.1| (SEQ ID NOS:122-126,respectively), which all have the function assigned to SEQ ID NO:120 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 9 depicts a sequence alignment between SEQ ID NO:137 (RAAC00632)and ref|YP_001126681.1|, ref|YP_148515.1|, ref|ZP_01171798.1|,ref|YP_001374758.1|, and ref|YP_080106.1| (SEQ ID NOS:139-143,respectively), which all have the function assigned to SEQ ID NO:137 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 10A and 10B depict a sequence alignment between SEQ ID NO:154(RAAC00633) and ref|NP_243928.1|, ref|ZP_01695378.1|,ref|ZP_01725506.1|, ref|YP_176142.1|, and ref|YP_850199.1| (SEQ IDNOS:156-160, respectively), which all have the function assigned to SEQID NO:154 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 11A and 11B depict a sequence alignment between SEQ ID NO:171(RAAC00634) and ref|YP_001126680.1|, ref|YP_001487695.1|,ref|YP_148514.11, gb|AAL99356.1|, and ref|YP_176141.1| (SEQ IDNOS:173-177, respectively), which all have the function assigned to SEQID NO:171 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 12 depicts a sequence alignment between SEQ ID NO:188 (RAAC00174)and ref|YP_175798.1|, ref|NP_243358.1|, ref|NP_389472.1|,ref|ZP_01861659.1|, and ref|YP_147042.1| (SEQ ID NOS:190-194,respectively), which all have the function assigned to SEQ ID NO:188 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 13 depicts a sequence alignment between SEQ ID NO:205 (RAAC00635)and ref|YP_148513.1|, ref|NP_243926.1|, ref|YP_001126679.1|,ref|YP_176140.1|, and ref|NP_843875.1| (SEQ ID NOS:207-211,respectively), which all have the function assigned to SEQ ID NO:205 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 14 depicts a sequence alignment between SEQ ID NO:222 (RAAC00637)and ref|NP_243923.1|, ref|YP_148510.1|, ref|ZP_01171803.1|,ref|YP_001126676.1|, and ref|NP_926497.1| (SEQ ID NOS:224-228,respectively), which all have the function assigned to SEQ ID NO:222 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 15A and 15B depict a sequence alignment between SEQ ID NO:239(RAAC00638) and ref|NP_243922.1|, ref|YP_148509.1|, ref|YP_001126675.1|,ref|ZP_01171804.1|, and ref|YP_075945.1| (SEQ ID NOS:241-245,respectively), which all have the function assigned to SEQ ID NO:239 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 16 depicts a sequence alignment between SEQ ID NO:256 (RAAC00639)and sp967MJ3|LEUD_SYMTH, ref|YP_148508.1|, ref|YP_001126674.1|,ref|YP_080099.1|, and ref|YP_001487689.1| (SEQ ID NOS:258-262,respectively), which all have the function assigned to SEQ ID NO:256 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 17A and 17B depict a sequence alignment between SEQ ID NO:273(RAAC00642) and ref|YP_826036.1|, gb|ABV27286.1|,gb|AAL17866.1|AF424980_1, ref|ZP_01859643.1|, and ref|NP_244026.1| (SEQID NOS:275-279, respectively), which all have the function assigned toSEQ ID NO:273 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 18A-18C depict a sequence alignment between SEQ ID NO:290(RAAC00727) and ref|YP_001637294.1|, ref|ZP_01516643.1|,ref|YP_645264.1|, ref|YP_146876.1|, and ref|YP_001125008.1| (SEQ IDNOS:292-296, respectively), which all have the function assigned to SEQID NO:290 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 19A and 19B depict a sequence alignment between SEQ ID NO:307(RAAC00729) and ref|YP_001125365.1|, ref|YP_147249.1|,ref|ZP_01695431.1|, ref|NP_244828.1|, and ref|YP_895448.1| (SEQ IDNOS:309-313, respectively), which all have the function assigned to SEQID NO:307 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 20A and 20B depict a sequence alignment between SEQ ID NO:324(RAAC00730) and ref|YP_075148.1|, sp|P164681MAOX_BACST,ref|YP_147293.1|, ref|YP_643888.1|, and ref|YP_001125416.1| (SEQ IDNOS:326-330, respectively), which all have the function assigned to SEQID NO:324 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 21A-21C depict a sequence alignment between SEQ ID NO:341(RAAC00735) and ref|ZP_01696337.1|, ref|ZP_02171753.1|,ref|YP_284976.1|, ref|YP_001546997.1|, and ref|YP_001277075.1| (SEQ IDNOS:343-347, respectively), which all have the function assigned to SEQID NO:341 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 22 depicts a sequence alignment between SEQ ID NO:358 (RAAC00812)and ref|ZP_00539373.1|, ref|YP_386234.1|, ref|YP_001378696.1|,ref|ZP_01723286.1|, and ref|NP_391778.1| (SEQ ID NOS:360-364,respectively), which all have the function assigned to SEQ ID NO:358 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 23A and 23B depict a sequence alignment between SEQ ID NO:375(RAAC00196) and ref|YP_147293.1|, sp|P16468|MAOX_BACST,ref|YP_643888.1|, ref|YP_075148.1|, and ref|YP_001125416.1| (SEQ IDNOS:377-381, respectively), which all have the function assigned to SEQID NO:375 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 24 depicts a sequence alignment between SEQ ID NO:392 (RAAC00814)and ref|YP_360188.1|, ref|ZP_01666093.1|, ref|NP_242895.1|,ref|YP_360122.1|, and ref|ZP_01372991.1| (SEQ ID NOS:394-398,respectively), which all have the function assigned to SEQ ID NO:392 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 25A and 25B depict a sequence alignment between SEQ ID NO:409(RAAC00815) and ref|YP_644483.1|, ref|NP_294183.1|, ref|YP_359514.1|,ref|YP_605214.1|, and ref|YP_592595.1| (SEQ ID NOS:411-415,respectively), which all have the function assigned to SEQ ID NO:409 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 26 depicts a sequence alignment between SEQ ID NO:426 (RAAC00816)and ref|YP_147450.1|, ref|YP_001125561.1|, ref|ZP_01696479.1|,ref|NP_241996.1|, and ref|YP_079308.1| (SEQ ID NOS:428-432,respectively), which all have the function assigned to SEQ ID NO:426 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 27 depicts a sequence alignment between SEQ ID NO:443 (RAAC00822)and ref|ZP_00539140.1|, ref|ZP_02130394.1|, ref|NP_241073.1|,ref|ZP_01696475.1|, and dbj|BAA75325.1| (SEQ ID NOS:445-449,respectively), which all have the function assigned to SEQ ID NO:443 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 28A-28C depict a sequence alignment between SEQ ID NO:460(RAAC00950) and ref|YP_001420821.1|, ref|ZP_01696606.1|,ref|ZP_01171726.11, ref|NP_389098.1|, and ref|YP_091797.1| (SEQ IDNOS:462-466, respectively), which all have the function assigned to SEQID NO:460 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 29 depicts a sequence alignment between SEQ ID NO:477 (RAAC00952)and ref|YP_146314.1|, ref|YP_001124593.1|, ref|NP_830405.1|,ref|ZP_00739906.1|, and ref|NP_391552.1| (SEQ ID NOS:479-483,respectively), which all have the function assigned to SEQ ID NO:477 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 30 depicts a sequence alignment between SEQ ID NO:494 (RAAC00990)and ref|YP_148038.1|, ref|YP_001126216.1|, ref|NP_242546.1|,ref|ZP_01697215.1|, and ref|YP_175412.1| (SEQ ID NOS:496-500,respectively), which all have the function assigned to SEQ ID NO:494 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 31 depicts a sequence alignment between SEQ ID NO:511 (RAAC01029)and ref|YP_001132791.1|, ref|YP_890165.1|, ref|YP_704478.1|,ref|YP_956012.1|, and ref|YP_879906.2| (SEQ ID NOS:513-517,respectively), which all have the function assigned to SEQ ID NO:511 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 32A-32C depict a sequence alignment between SEQ ID NO:528(RAAC01041) and ref|YP_359304.1|, ref|ZP_01697277.1|, ref|YP_519313.1|,ref|ZP_01370069.1|, and ref|YP_429480.1| (SEQ ID NOS:530-534,respectively), which all have the function assigned to SEQ ID NO:528 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 33A and 33B depict a sequence alignment between SEQ ID NO:545(RAAC01057) and ref|YP_148861.1|, ref|YP_076839.1|, ref|NP_244355.1|,ref|ZP_01697463.1|, and ref|ZP_01173543.1| (SEQ ID NOS:547-551,respectively), which all have the function assigned to SEQ ID NO:545 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 34 depicts a sequence alignment between SEQ ID NO:562 (RAAC00352)and ref|NP_691707.1|, ref|YP_829756.1|, ref|YP_947785.1|,ref|YP_001221402.1|, and ref|YP_885435.1| (SEQ ID NOS:564-568,respectively), which all have the function assigned to SEQ ID NO:562 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 35 depicts a sequence alignment between SEQ ID NO:579 (RAAC04321)and gb|ABW71834.1|, ref|YP_055250.1|, ref|YP_612035.1|,ref|YP_134751.1|, and ref|ZP_01441442.1| (SEQ ID NOS:581-585,respectively), which all have the function assigned to SEQ ID NO:579 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 36 depicts a sequence alignment between SEQ ID NO:596 (RAAC04349)and ref|YP_917551.1|, ref|ZP_00631342.1|, ref|YP_001259911.1|,ref|NP_105797.1|, and ref|ZP_00998521.1 (SEQ ID NOS:598-602,respectively), which all have the function assigned to SEQ ID NO:596 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 37A and 37B depict a sequence alignment between SEQ ID NO:613(RAAC01327) and emb|CAD30313.1|, ref|ZP_01697379.1|,ref|YP_001375474.11, ref|NP_833288.1|, and ref|NP_979866.1| (SEQ IDNOS:615-619, respectively), which all have the function assigned to SEQID NO:613 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 38A and 38B depict a sequence alignment between SEQ ID NO:630(RAAC01351) and ref|YP_001125497.1|, ref|YP_175672.1|, ref|NP_243001.1|,ref|YP_147384.11, and ref|YP_001108459.1| (SEQ ID NOS:632-636,respectively), which all have the function assigned to SEQ ID NO:630 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 39A and 39B depict a sequence alignment between SEQ ID NO:647(RAAC01352) and ref|YP_147385.1|, ref|YP_001125498.1|, ref|YP_175671.1|,ref|NP_926015.11, and ref|YP_001660274.1| (SEQ ID NOS:649-653,respectively), which all have the function assigned to SEQ ID NO:647 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 40A and 40B depict a sequence alignment between SEQ ID NO:664(RAAC01354) and ref|YP_001636557.1|, ref|ZP_01517435.1|,ref|ZP_01697170.1|, ref|YP_001374183.1|, and ref|YP_082630.1| (SEQ IDNOS:666-670, respectively), which all have the function assigned to SEQID NO:664 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 41A and 41B depict a sequence alignment between SEQ ID NO:681(RAAC01360) and ref|ZP_01724857.1|, ref|ZP_00235684.1|,ref|YP_895924.1|, ref|YP_037600.1|, and ref|YP_001646030.1| (SEQ IDNOS:683-687, respectively), which all have the function assigned to SEQID NO:681 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 42 depicts a sequence alignment between SEQ ID NO:698 (RAAC01408)and ref|YP_872951.1|, gb|AAQ84159.1|, ref|YP_701593.1|,ref|YP_885121.1|, and ref|ZP_02169377.1| (SEQ ID NOS:700-704,respectively), which all have the function assigned to SEQ ID NO:698 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 43 depicts a sequence alignment between SEQ ID NO:715 (RAAC01425)and ref|YP_146050.1|, ref|YP_001124307.1|, ref|YP_360564.1|,ref|NP_691609.1|, and ref|NP_294646.1| (SEQ ID NOS:717-721,respectively), which all have the function assigned to SEQ ID NO:715 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 44 depicts a sequence alignment between SEQ ID NO:732 (RAAC01517)and ref|YP_902570.1|, ref|YP_076319.1|, ref|YP_001629366.1|,ref|ZP_01667660.1|, and ref|YP_429281.1| (SEQ ID NOS:734-738,respectively), which all have the function assigned to SEQ ID NO:732 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 45 depicts a sequence alignment between SEQ ID NO:749 (RAAC00449)and ref|ZP_01666747.1|, pdb|2QE7|H, sp|P22480|ATPE_BACPF,ref|ZP_01188594.1|, and ref|YP_521144.1| (SEQ ID NOS:751-755,respectively), which all have the function assigned to SEQ ID NO:749 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 46A and 46B depict a sequence alignment between SEQ ID NO:766(RAAC01555) and ref|YP_079644.1|, sp|P23630|DCDA_BACSU,ref|NP_390219.1|, ref|YP_001421740.1|, and ref|YP_001487298.1| (SEQ IDNOS:768-772, respectively), which all have the function assigned to SEQID NO:766 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 47A and 47B depict a sequence alignment between SEQ ID NO:783(RAAC01575) and ref|NP_241871.1|, ref|YP_077980.1|, ref|YP_001420375.1|,ref|NP_388616.1|, and ref|NP_693628.1| (SEQ ID NOS:785-789,respectively), which all have the function assigned to SEQ ID NO:783 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 48 depicts a sequence alignment between SEQ ID NO:800 (RAAC01657)and dbj|BAB40585.1|, ref|NP_241079.1|, ref|YP_001126012.1|,ref|ZP_01171269.1|, and ref|ZP_01860561.1| (SEQ ID NOS:802-806,respectively), which all have the function assigned to SEQ ID NO:800 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 49 depicts a sequence alignment between SEQ ID NO:817 (RAAC01658)and ref|NP_241080.1|, dbj|BAB40586.1|, ref|YP_001126011.1|,ref|NP_693798.1|, and ref|ZP_00539126.1| (SEQ ID NOS:819-823,respectively), which all have the function assigned to SEQ ID NO:817 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 50 depicts a sequence alignment between SEQ ID NO:834 (RAAC01669)and ref|YP_001125402.1|, ref|YP_147282.1|, ref|ZP_01859257.1|,ref|NP_388913.1|, and ref|YP_001420249.1| (SEQ ID NOS:836-840,respectively), which all have the function assigned to SEQ ID NO:834 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 51A-51C depict a sequence alignment between SEQ ID NO:851(RAAC01678) and ref|ZP_01696606.1|, ref|YP_146312.1|,ref|ZP_01171726.11, ref|YP_001124591.1|, and ref|ZP_01696079.1| (SEQ IDNOS:853-857, respectively), which all have the function assigned to SEQID NO:851 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 52A and 52B depict a sequence alignment between SEQ ID NO:868(RAAC01685) and ref|YP_431081.1|, ref|YP_001211085.1|,ref|YP_001111663.11, ref|YP_001547204.1|, and ref|NP_213242.1| (SEQ IDNOS:870-874, respectively), which all have the function assigned to SEQID NO:868 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 53 depicts a sequence alignment between SEQ ID NO:885 (RAAC01745)and ref|YP_001127228.1|, ref|YP_149070.1|, ref|ZP_00539127.1|,ref|NP_241079.1|, and ref|YP_074240.1| (SEQ ID NOS:887-891,respectively), which all have the function assigned to SEQ ID NO:885 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 54 depicts a sequence alignment between SEQ ID NO:902 (RAAC01746)and ref|YP_149069.1|, ref|YP_001127227.1|, ref|ZP_00539126.1|,ref|YP_001125046.1|, and ref|NP_833691.1| (SEQ ID NOS:904-908,respectively), which all have the function assigned to SEQ ID NO:902 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 55 depicts a sequence alignment between SEQ ID NO:919 (RAAC01748)and ref|NP_828658.1|, emb|CAJ88521.1|, ref|NP_625066.1|,ref|YP_001104836.1|, and ref|YP_658557.1| (SEQ ID NOS:921-925,respectively), which all have the function assigned to SEQ ID NO:919 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 56A and 56B depict a sequence alignment between SEQ ID NO:936(RAAC00450) and pdb|2QE7|D, sp|Q9LA80|ATPB_GEOTH, ref|YP_149211.11,sp|P41009|ATPB_BACCA, and prf∥1211283A (SEQ ID NOS:938-942,respectively), which all have the function assigned to SEQ ID NO:936 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 57A-57C depict a sequence alignment between SEQ ID NO:953(RAAC01759) and ref|ZP_02170376.1|, ref|YP_001546865.1|,ref|YP_001125323.1|, ref|YP_147200.2|, and ref|YP_091630.1| (SEQ IDNOS:955-959, respectively), which all have the function assigned to SEQID NO:953 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 58 depicts a sequence alignment between SEQ ID NO:970 (RAAC01762)and gb|ABW71834.1|, ref|ZP_02015336.1|, ref|YP_055250.1|,ref|YP_136548.1|, and ref|NP_102793.1| (SEQ ID NOS:972-976,respectively), which all have the function assigned to SEQ ID NO:970 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 59 depicts a sequence alignment between SEQ ID NO:987 (RAAC01763)and ref|YP_300327.1|, ref|NP_693723.1|, ref|YP_190012.1|,ref|YP_252288.1|, and ref|ZP_01227084.1| (SEQ ID NOS:989-993,respectively), which all have the function assigned to SEQ ID NO:987 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 60 depicts a sequence alignment between SEQ ID NO:1004 (RAAC01767)and ref|ZP_01860323.1|, ref|NP_244450.1|, ref|YP_148929.1|,ref|YP_080823.1|, and ref|ZP_01171654.1| (SEQ ID NOS:1006-1010,respectively), which all have the function assigned to SEQ ID NO:1004 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 61 depicts a sequence alignment between SEQ ID NO:1021 (RAAC01797)and ref|YP_076186.1|, ref|NP_691214.1|, ref|ZP_01170331.1|,ref|NP_388333.1|, and ref|YP_001375327.1| (SEQ ID NOS:1023-1027,respectively), which all have the function assigned to SEQ ID NO:1021 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 62A and 62B depict a sequence alignment between SEQ ID NO:1038(RAAC01900) and ref|NP_691405.1|, ref|NP_242876.1|, ref|NP_241871.1|,ref|YP_001420375.11, and ref|NP_388616.1| (SEQ ID NOS:1040-1044,respectively), which all have the function assigned to SEQ ID NO:1038 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 63 depicts a sequence alignment between SEQ ID NO:1055 (RAAC01939)and ref|NP_390790.1|, ref|ZP_02170616.1|, ref|NP_693087.1|,ref|YP_080204.1|, and sp|Q59202|MDH_BACIS (SEQ ID NOS:1057-1061,respectively), which all have the function assigned to SEQ ID NO:1055 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 64 depicts a sequence alignment between SEQ ID NO:1072 (RAAC01996)and ref|NP_244279.1|, ref|YP_001126997.1|, ref|YP_148810.1|,ref|YP_001488092.1|, and ref|NP_391097.1| (SEQ ID NOS:1074-1078,respectively), which all have the function assigned to SEQ ID NO:1072Table 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 65 depicts a sequence alignment between SEQ ID NO:1089 (RAAC02025)and ref|NP_390723.1|, ref|YP_080139.1|, ref|YP_001422141.1|,ref|ZP_01171785.1|, and ref|NP_243959.1| (SEQ ID NOS:1091-1095,respectively), which all have the function assigned to SEQ ID NO:1089 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 66 depicts a sequence alignment between SEQ ID NO:1106 (RAAC00451)and pdb|2QE7|G, ref|YP_001127389.1|, ref|YP_149212.1|,ref|YP_001488540.1|, and emb|CAA30654.1| (SEQ ID NOS:1108-1112,respectively), which all have the function assigned to SEQ ID NO:1106 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 67A and 67B depict a sequence alignment between SEQ ID NO:1123(RAAC02026) and ref|YP_148525.1|, ref|YP_001126690.1|, emb|CAA69872.1|,ref|YP_092553.11, and ref|NP_243958.1| (SEQ ID NOS:1125-1129,respectively), which all have the function assigned to SEQ ID NO:1123 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 68 depicts a sequence alignment between SEQ ID NO:1140 (RAAC02027)and emb|CAA69873.1|, ref|YP_148524.1|, ref|YP_080136.1|,ref|NP_243957.1|, and ref|ZP_01697535.1| (SEQ ID NOS:1142-1146,respectively), which all have the function assigned to SEQ ID NO:1140 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 69 depicts a sequence alignment between SEQ ID NO:1157 (RAAC02040)and ref|ZP_01697399.1|, ref|YP_001124579.1|, ref|YP_146298.1|,ref|NP_691785.1|, and ref|ZP_01723229.1| (SEQ ID NOS:1159-1163,respectively), which all have the function assigned to SEQ ID NO:1157 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 70A and 70B depict a sequence alignment between SEQ ID NO:1174(RAAC02181) and ref|NP_391000.1|, ref|YP_080655.1|, ref|YP_173878.1|,ref|NP_242416.1|, and ref|YP_644452.1| (SEQ ID NOS:1176-1180,respectively), which all have the function assigned to SEQ ID NO:1174 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 71 depicts a sequence alignment between SEQ ID NO:1191 (RAAC02222)and ref|YP_001487576.1|, ref|ZP_02211990.1|, ref|YP_001343716.1|,ref|NP_744947.1|, and ref|YP_633768.1| (SEQ ID NOS:1193-1197,respectively), which all have the function assigned to SEQ ID NO:1191 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 72A and 72B depict a sequence alignment between SEQ ID NO:1208(RAAC02274) and ref|YP_146053.1|, ref|ZP_01869175.1|,ref|ZP_00989613.11, ref|YP_001276414.1|, and ref|YP_001211401.1| (SEQ IDNOS:1210-1214, respectively), which all have the function assigned toSEQ ID NO:1208 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 73A and 73B depict a sequence alignment between SEQ ID NO:1225(RAAC02275) and ref|YP_146052.1|, ref|YP_001546552.1|,ref|YP_001636911.11, ref|ZP_01514632.1|, and ref|YP_001274650.1| (SEQ IDNOS:1227-1231, respectively), which all have the function assigned toSEQ ID NO:1225 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 74 depicts a sequence alignment between SEQ ID NO:1242 (RAAC02426)and ref|NP_243521.1|, pdb|1W851A, sp1P21873|ODPA_BACST,ref|YP_001421036.1|, and ref|YP_146911.1| (SEQ ID NOS:1244-1248,respectively), which all have the function assigned to SEQ ID NO:1242 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 75 depicts a sequence alignment between SEQ ID NO:1259 (RAAC02427)and ref|ZP_01696304.1|, sp|P21874|ODPB_BACST, ref|YP_001125046.1|,pdb|1W85|B, and ref|YP_146912.1| (SEQ ID NOS:1261-1265, respectively),which all have the function assigned to SEQ ID NO:1259 Table 1. Aminoacids conserved among all sequences are indicted by a “*” and generallyconserved amino acids are indicated by a “:”.

FIGS. 76A and 76B depict a sequence alignment between SEQ ID NO:1276(RAAC02429) and ref|YP_001125048.1|, sp|P11959|DLDH1_BACST,ref|YP_146914.11, ref|YP_001486601.1|, and pdb|1EBD|A (SEQ IDNOS:1278-1282, respectively), which all have the function assigned toSEQ ID NO:1276 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 77A and 77B depicts a sequence alignment between SEQ ID NO:1293(RAAC00452) and pdb|2QE7|A, ref|YP_361340.1|, ref|YP_001127390.1|,ref|YP_149213.1|, and ref|YP_001356688.1| (SEQ ID NOS:1295-1299,respectively), which all have the function assigned to SEQ ID NO:1293 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 78 depicts a sequence alignment between SEQ ID NO:1310 (RAAC02433)and ref|YP_001542913.1|, ref|YP_644829.1|, ref|YP_356005.1|,emb|CA090974.1|, and ref|YP_001656571.1| (SEQ ID NOS:1312-1316,respectively), which all have the function assigned to SEQ ID NO:1310 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 79A and 79B depict a sequence alignment between SEQ ID NO:1327(RAAC02438) and ref|YP_644476.1|, ref|ZP_02191297.1|,ref|ZP_01549387.11, ref|ZP_01850519.1|, and ref|ZP_01015586.1| (SEQ IDNOS:1329-1333, respectively), which all have the function assigned toSEQ ID NO:1327 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 80A and 80B depict a sequence alignment between SEQ ID NO:1344(RAAC02441) and ref|YP_147804.1|, ref|YP_001125954.1|,ref|YP_001125911.1|, ref|YP_147740.1|, and ref|NP_243178.1| (SEQ IDNOS:1346-1350, respectively), which all have the function assigned toSEQ ID NO:1344 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 81 depicts a sequence alignment between SEQ ID NO:1361 (RAAC02442)and ref|YP_001125956.1|, ref|YP_147805.1|, ref|ZP_01169177.1|,ref|ZP_01695873.1|, and ref|NP_831941.1| (SEQ ID NOS:1363-1367,respectively), which all have the function assigned to SEQ ID NO:1361 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 82A and 82B depict a sequence alignment between SEQ ID NO:1378(RAAC02630) and ref|ZP_01695367.1|, ref|YP_723673.1|, ref|YP_686117.1|,ref|YP_001111391.1|, and ref|ZP_01623360.1| (SEQ ID NOS:1380-1384,respectively), which all have the function assigned to SEQ ID NO:1378 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 83 depicts a sequence alignment between SEQ ID NO:1395 (RAAC02644)and ref|NP_782567.1|, sp9892U0|LDH_CLOTE, ref|YP_590559.1|,ref|ZP_01514103.1|, and ref|YP_009822.1| (SEQ ID NOS:1397-1401,respectively), which all have the function assigned to SEQ ID NO:1395 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 84A and 84B depict a sequence alignment between SEQ ID NO:1412(RAAC02702) and ref|YP_001124710.1|, ref|YP_146529.1|, ref|NP_977551.1|,ref|YP_893868.1|, and ref|NP_843617.1| (SEQ ID NOS:1414-1418,respectively), which all have the function assigned to SEQ ID NO:1412 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 85A and 85B depict a sequence alignment between SEQ ID NO:1429(RAAC04058) and ref|ZP_02080303.1|, ref|YP_520543.1|,ref|ZP_01966380.11, ref|ZP_02039587.1|, and ref|ZP_02073747.1| (SEQ IDNOS:1431-1435, respectively), which all have the function assigned toSEQ ID NO:1429 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 86A and 86B depict a sequence alignment between SEQ ID NO:1446(RAAC02843) and ref|YP_001125182.1|, ref|YP_147061.1|,ref|ZP_01171540.1|, ref|NP_692464.1|, and ref|YP_001375719.1| (SEQ IDNOS:1448-1452, respectively), which all have the function assigned toSEQ ID NO:1446 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 87 depicts a sequence alignment between SEQ ID NO:1463 (RAAC02844)and ref|YP_147062.1|, ref|YP_001125183.1|, ref|YP_079003.1|,ref|NP_243335.1|, and ref|ZP_01171539.1| (SEQ ID NOS:1465-1469,respectively), which all have the function assigned to SEQ ID NO:1463 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 88 depicts a sequence alignment between SEQ ID NO:1480 (RAAC00454)and ref|YP_001127392.1|, ref|YP_521149.1|, ref|YP_149215.1|,ref|YP_001488543.1|, and ref|YP_093437.1| (SEQ ID NOS:1482-1486,respectively), which all have the function assigned to SEQ ID NO:1480 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIG. 89 depicts a sequence alignment between SEQ ID NO:1497 (RAAC02920)and ref|YP_001421255.1|, ref|NP_389559.1|, ref|YP_001125250.1|,ref|ZP_02169638.1|, and ref|YP_091490.1| (SEQ ID NOS:1499-1503,respectively), which all have the function assigned to SEQ ID NO:1497 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 90A and 90B depict a sequence alignment between SEQ ID NO:1514(RAAC02924) and ref|YP_001375026.1|, ref|YP_175305.1|, ref|NP_844736.1|,ref|NP_691737.1|, and ref|NP_832053.1| (SEQ ID NOS:1516-1520,respectively), which all have the function assigned to SEQ ID NO:1514 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

FIGS. 91A and 91B depict a sequence alignment between SEQ ID NO:1531(RAAC02926) and ref|YP_148646.1|, ref|ZP_01696063.1|,ref|ZP_01859600.1|, ref|YP_001376529.1|, and ref|YP_038694.1| (SEQ IDNOS:1533-1537, respectively), which all have the function assigned toSEQ ID NO:1531 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIGS. 92A and 92B depict a sequence alignment between SEQ ID NO:1548(RAAC02986) and ref|YP_146227.1|, ref|YP_001124476.1|,ref|ZP_01695767.1|, dbj|BAB39706.1|, and ref|ZP_01723231.1| (SEQ IDNOS:1550-1554, respectively), which all have the function assigned toSEQ ID NO:1548 in Table 1. Amino acids conserved among all sequences areindicted by a “*” and generally conserved amino acids are indicated by a“:”.

FIG. 93 depicts a sequence alignment between SEQ ID NO:1565 (RAAC03010)and ref|YP_001127080.1|, ref|YP_148885.1|, ref|YP_001374290.1|,ref|NP_693789.1|, and ref|YP_144223.1| (SEQ ID NOS:1567-1571,respectively), which all have the function assigned to SEQ ID NO:1565 inTable 1. Amino acids conserved among all sequences are indicted by a “*”and generally conserved amino acids are indicated by a “:”.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention include genes and associated proteinsrelated to the metabolism of the thermoacidophile Alicyclobacillusacidocaldarius. Coding sequences for genes related to these processeswere determined from sequence information generated from sequencing thegenome of Alicyclobacillus acidocaldarius. These genes and proteins mayrepresent targets and/or elements of transformation systems or vectorsfor metabolic engineering of Alicyclobacillus acidocaldarius or otherorganisms. Non-limiting examples of nucleotide sequences found withinthe genome of Alicyclobacillus acidocaldarius, and amino acids codedthereby, associated with metabolism are listed in Table 1. Metabolismproteins may be, without limitation, of the following classes:(S)-2-hydroxy-acid oxidases, [acyl-carrier-protein]S-malonyltransferases, 1,3-propanediol Dehydrogenases, 2-isopropylmalateSynthases, 3-hydroxybutyryl-CoA dehydratases, 3-isopropylmalateDehydratases, 3-isopropylmalate Dehydrogenases, 3-oxoacidCoA-transferases, 8-amino-7-oxononanoate Synthases, Acetaldehydedehydrogenases (acetylating), Acetate-CoA ligases, Acetolactatesynthases, Acetyl-CoA C-acetyltransferases, Aconitate hydratases,Alcohol dehydrogenases, Alcohol dehydrogenases (NADP+), Aldehydedehydrogenases, Aldehyde dehydrogenases (NAD+), ATPphosphoribosyltransferases, ATP synthase alpha chains, ATP synthase Bchains, ATP synthase beta chains, ATP synthase C chains, ATP synthaseepsilon chains, ATP synthase gamma chains, Biotin synthases,Branched-chain-amino-acid transaminases, Butyryl-CoA dehydrogenases,Citrate (Si)-synthases, Dethiobiotin synthases, Diaminopimelatedecarboxylases, Diaminopimelate epimerases, Dihydrodipicolinatereductases, Dihydrodipicolinate synthases, Dihydrolipoyl dehydrogenases,Dihydroxy-acid dehydratases, Enoyl-CoA hydratases, FdhD proteins (fdsC),Formate dehydrogenases, Glycerate kinases, Glycinehydroxymethyltransferases, Isocitrate lyases, Lactaldehyde reductases,Lactate 2-monooxygenases, L-lactate dehydrogenases, Malatedehydrogenases, Malate dehydrogenases (acceptor), Malate dehydrogenases(oxaloacetate-decarboxylating), Malate synthases, Malonate-semialdehydedehydrogenases (acetylating), Methylmalonate-semialdehyde dehydrogenases(acylating), N-acetyldiaminopimelate deacetylases, Oxoglutaratedehydrogenases (succinyl-transferring), Phosphoenolpyruvatecarboxylases, Phosphoglycerate dehydrogenases,Phosphoribosylanthranilate isomerases, Pyruvate dehydrogenases(acetyl-transferring), Pyruvate phosphate dikinases, Succinatedehydrogenase cytochrome b558 subunits, Succinate dehydrogenaseflavoprotein subunits, Succinate dehydrogenase iron-sulfur proteins,Succinate-CoA ligases (ADP-forming); and others.

Embodiments of the invention relate in part to the gene sequences and/orprotein sequences comprising genes and/or proteins of Alicyclobacillusacidocaldarius. Genes and proteins included are those that play a rolein metabolism. Intracellular enzyme activities may be thermophilicand/or acidophilic in nature and general examples of similar genes aredescribed in the literature. Classes of genes, sequences, enzymes andfactors include, but are not limited to, those listed in Table 1.

TABLE 1 Alicyclobacillus acidocaldarius genes related to metabolismReference Gene Sequence Protein Sequence Function RAAC00079 SEQ ID NO: 1SEQ ID NO: 2 Acetate-CoA ligase RAAC00455 SEQ ID NO: 18 SEQ ID NO: 19ATP synthase C chain RAAC00461 SEQ ID NO: 35 SEQ ID NO: 36 Glycinehydroxymethyltransferase RAAC00481 SEQ ID NO: 52 SEQ ID NO: 533-hydroxybutyryl-CoA dehydratase RAAC00529 SEQ ID NO: 69 SEQ ID NO: 70N-acetyldiaminopimelate deacetylase RAAC00552 SEQ ID NO: 86 SEQ ID NO:87 Biotin synthase RAAC00553 SEQ ID NO: 103 SEQ ID NO: 104 Dethiobiotinsynthase RAAC00554 SEQ ID NO: 120 SEQ ID NO: 121 8-amino-7-oxononanoateSynthase RAAC00632 SEQ ID NO: 137 SEQ ID NO: 138Branched-chain-amino-acid transaminase RAAC00633 SEQ ID NO: 154 SEQ IDNO: 155 Dihydroxy-acid dehydratase RAAC00634 SEQ ID NO: 171 SEQ ID NO:172 Acetolactate synthase RAAC00174 SEQ ID NO: 188 SEQ ID NO: 189[acyl-carrier-protein] S-malonyltransferase RAAC00635 SEQ ID NO: 205 SEQID NO: 206 Acetolactate synthase RAAC00637 SEQ ID NO: 222 SEQ ID NO: 2233-isopropylmalate Dehydrogenase RAAC00638 SEQ ID NO: 239 SEQ ID NO: 2403-isopropylmalate Dehydratase RAAC00639 SEQ ID NO: 256 SEQ ID NO: 2573-isopropylmalate Dehydratase RAAC00642 SEQ ID NO: 273 SEQ ID NO: 274Citrate (Si)-synthase RAAC00727 SEQ ID NO: 290 SEQ ID NO: 291Oxoglutarate dehydrogenase (succinyl-transferring) RAAC00729 SEQ ID NO:307 SEQ ID NO: 308 Malate dehydrogenase (acceptor) RAAC00730 SEQ ID NO:324 SEQ ID NO: 325 Malate dehydrogenase (oxaloacetate-decarboxylating)RAAC00735 SEQ ID NO: 341 SEQ ID NO: 342 Phosphoenolpyruvate carboxylaseRAAC00812 SEQ ID NO: 358 SEQ ID NO: 359 3-oxoacid CoA-transferaseRAAC00196 SEQ ID NO: 375 SEQ ID NO: 376 Malate dehydrogenase(oxaloacetate-decarboxylating) RAAC00814 SEQ ID NO: 392 SEQ ID NO: 393Acetyl-CoA C-acetyltransferase RAAC00815 SEQ ID NO: 409 SEQ ID NO: 410Acetate-CoA ligase RAAC00816 SEQ ID NO: 426 SEQ ID NO: 427 Butyryl-CoAdehydrogenase RAAC00822 SEQ ID NO: 443 SEQ ID NO: 4443-hydroxybutyryl-CoA dehydratase RAAC00950 SEQ ID NO: 460 SEQ ID NO: 461Formate dehydrogenase RAAC00952 SEQ ID NO: 477 SEQ ID NO: 478 FdhDprotein (fdsC) RAAC00990 SEQ ID NO: 494 SEQ ID NO: 495Dihydrodipicolinate reductase RAAC01029 SEQ ID NO: 511 SEQ ID NO: 512Acetaldehyde dehydrogenase (acetylating) RAAC01041 SEQ ID NO: 528 SEQ IDNO: 529 Pyruvate, phosphate dikinase RAAC01057 SEQ ID NO: 545 SEQ ID NO:546 Enoyl-CoA hydratase RAAC00352 SEQ ID NO: 562 SEQ ID NO: 563 Alcoholdehydrogenase (NADP+) RAAC04321 SEQ ID NO: 579 SEQ ID NO: 580 Alcoholdehydrogenase RAAC04349 SEQ ID NO: 596 SEQ ID NO: 597Phosphoribosylanthranilate isomerase RAAC01327 SEQ ID NO: 613 SEQ ID NO:614 Aldehyde dehydrogenase (NAD+) RAAC01351 SEQ ID NO: 630 SEQ ID NO:631 (S)-2-hydroxy-acid oxidase RAAC01352 SEQ ID NO: 647 SEQ ID NO: 648(S)-2-hydroxy-acid oxidase RAAC01354 SEQ ID NO: 664 SEQ ID NO: 665Malate synthase RAAC01360 SEQ ID NO: 681 SEQ ID NO: 682(S)-2-hydroxy-acid oxidase RAAC01408 SEQ ID NO: 698 SEQ ID NO: 699Butyryl-CoA dehydrogenase RAAC01425 SEQ ID NO: 715 SEQ ID NO: 716Butyryl-CoA dehydrogenase RAAC01517 SEQ ID NO: 732 SEQ ID NO: 733Glycerate kinase RAAC00449 SEQ ID NO: 749 SEQ ID NO: 750 ATP synthaseepsilon chain RAAC01555 SEQ ID NO: 766 SEQ ID NO: 767 Diaminopimelatedecarboxylase RAAC01575 SEQ ID NO: 783 SEQ ID NO: 784 Aldehydedehydrogenase (NAD+) RAAC01657 SEQ ID NO: 800 SEQ ID NO: 801 Pyruvatedehydrogenase (acetyl-transferring) RAAC01658 SEQ ID NO: 817 SEQ ID NO:818 Pyruvate dehydrogenase (acetyl-transferring) RAAC01669 SEQ ID NO:834 SEQ ID NO: 835 Alcohol dehydrogenase RAAC01678 SEQ ID NO: 851 SEQ IDNO: 852 Formate dehydrogenase RAAC01685 SEQ ID NO: 868 SEQ ID NO: 8692-isopropylmalate Synthase RAAC01745 SEQ ID NO: 885 SEQ ID NO: 886Pyruvate dehydrogenase (acetyl-transferring) RAAC01746 SEQ ID NO: 902SEQ ID NO: 903 Pyruvate dehydrogenase (acetyl-transferring) RAAC01748SEQ ID NO: 919 SEQ ID NO: 920 Lactate 2-monooxygenase RAAC00450 SEQ IDNO: 936 SEQ ID NO: 937 ATP synthase beta chain RAAC01759 SEQ ID NO: 953SEQ ID NO: 954 Aconitate hydratase RAAC01762 SEQ ID NO: 970 SEQ ID NO:971 Alcohol dehydrogenase RAAC01763 SEQ ID NO: 987 SEQ ID NO: 988Alcohol dehydrogenase RAAC01767 SEQ ID NO: 1004 SEQ ID NO: 1005 ATPphosphoribosyltransferase RAAC01797 SEQ ID NO: 1021 SEQ ID NO: 1022Butyryl-CoA dehydrogenase RAAC01900 SEQ ID NO: 1038 SEQ ID NO: 1039Aldehyde dehydrogenase RAAC01939 SEQ ID NO: 1055 SEQ ID NO: 1056 Malatedehydrogenase RAAC01996 SEQ ID NO: 1072 SEQ ID NO: 1073 Diaminopimelateepimerase RAAC02025 SEQ ID NO: 1089 SEQ ID NO: 1090 Succinatedehydrogenase cytochrome b558 subunit RAAC00451 SEQ ID NO: 1106 SEQ IDNO: 1107 ATP synthase gamma chain RAAC02026 SEQ ID NO: 1123 SEQ ID NO:1124 Succinate dehydrogenase flavoprotein subunit RAAC02027 SEQ ID NO:1140 SEQ ID NO: 1141 Succinate dehydrogenase iron-sulfur proteinRAAC02040 SEQ ID NO: 1157 SEQ ID NO: 1158 Butyryl-CoA dehydrogenaseRAAC02181 SEQ ID NO: 1174 SEQ ID NO: 1175 Lactaldehyde reductaseRAAC02222 SEQ ID NO: 1191 SEQ ID NO: 1192 1,3-propanediol DehydrogenaseRAAC02274 SEQ ID NO: 1208 SEQ ID NO: 1209 Alcohol dehydrogenaseRAAC02275 SEQ ID NO: 1225 SEQ ID NO: 1226 Aldehyde dehydrogenase (NAD+)RAAC02426 SEQ ID NO: 1242 SEQ ID NO: 1243 Pyruvate dehydrogenase(acetyl-transferring) RAAC02427 SEQ ID NO: 1259 SEQ ID NO: 1260 Pyruvatedehydrogenase (acetyl-transferring) RAAC02429 SEQ ID NO: 1276 SEQ ID NO:1277 Dihydrolipoyl dehydrogenase RAAC00452 SEQ ID NO: 1293 SEQ ID NO:1294 ATP synthase alpha chain RAAC02433 SEQ ID NO: 1310 SEQ ID NO: 13113-isopropylmalate Dehydrogenase RAAC02438 SEQ ID NO: 1327 SEQ ID NO:1328 Acetate-CoA ligase RAAC02441 SEQ ID NO: 1344 SEQ ID NO: 1345Malonate-semialdehyde dehydrogenase (acetylating) RAAC02442 SEQ ID NO:1361 SEQ ID NO: 1362 1,3-propanediol Dehydrogenase RAAC02630 SEQ ID NO:1378 SEQ ID NO: 1379 Phosphoglycerate dehydrogenase RAAC02644 SEQ ID NO:1395 SEQ ID NO: 1396 L-lactate dehydrogenase RAAC02702 SEQ ID NO: 1412SEQ ID NO: 1413 Isocitrate lyase RAAC04058 SEQ ID NO: 1429 SEQ ID NO:1430 2-isopropylmalate Synthase RAAC02843 SEQ ID NO: 1446 SEQ ID NO:1447 Succinate-CoA ligase (ADP-forming) RAAC02844 SEQ ID NO: 1463 SEQ IDNO: 1464 Succinate-CoA ligase (ADP-forming) RAAC00454 SEQ ID NO: 1480SEQ ID NO: 1481 ATP synthase B chain RAAC02920 SEQ ID NO: 1497 SEQ IDNO: 1498 Dihydrodipicolinate synthase RAAC02924 SEQ ID NO: 1514 SEQ IDNO: 1515 Methylmalonate-semialdehyde dehydrogenase (acylating) RAAC02926SEQ ID NO: 1531 SEQ ID NO: 1532 Acetate-CoA ligase RAAC02986 SEQ ID NO:1548 SEQ ID NO: 1549 Aldehyde dehydrogenase (NAD+) RAAC03010 SEQ ID NO:1565 SEQ ID NO: 1566 Dihydrodipicolinate synthase

The present invention relates to nucleotides sequences comprisingisolated and/or purified nucleotide sequences of the genome ofAlicyclobacillus acidocaldarius selected from the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954,971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158,1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362,1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and1566 or one of their fragments.

The present invention likewise relates to isolated and/or purifiednucleotide sequences, characterized in that they comprise at least oneof: a) a nucleotide sequence of at least one of the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954,971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158,1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362,1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and1566 or one of their fragments; b) a nucleotide sequence homologous to anucleotide sequence such as defined in a); c) a nucleotide sequencecomplementary to a nucleotide sequence such as defined in a) or b), anda nucleotide sequence of their corresponding RNA; d) a nucleotidesequence capable of hybridizing under stringent conditions with asequence such as defined in a), b) or c); e) a nucleotide sequencecomprising a sequence such as defined in a), b), c) or d); and f) anucleotide sequence modified by a nucleotide sequence such as defined ina), b), c), d) or e).

Nucleotide, polynucleotide, or nucleic acid sequence will be understoodaccording to the present invention as meaning both a double-stranded orsingle-stranded DNA in the monomeric and dimeric (so-called in tandem)forms and the transcription products of the DNAs.

Aspects of the invention relate to nucleotide sequences in which it hasbeen possible to isolate, purify or partially purify, starting fromseparation methods such as, for example, ion-exchange chromatography, byexclusion based on molecular size, or by affinity, or, alternatively,fractionation techniques based on solubility in different solvents, orstarting from methods of genetic engineering such as amplification,cloning, and subcloning, it being possible for the sequences of theinvention to be carried by vectors.

Isolated and/or purified nucleotide sequence fragment according to theinvention will be understood as designating any nucleotide fragment ofthe genome of Alicyclobacillus acidocaldarius, and may include, by wayof non-limiting example, a length of at least 8, 12, 20 25, 50, 75, 100,200, 300, 400, 500, 1000, or more, consecutive nucleotides of thesequence from which it originates.

Specific fragment of an isolated and/or purified nucleotide sequenceaccording to the invention will be understood as designating anynucleotide fragment of the genome of Alicyclobacillus acidocaldarius,having, after alignment and comparison with the corresponding fragmentsof genomic sequences of Alicyclobacillus acidocaldarius, at least onenucleotide or base of different nature.

Homologous isolated and/or purified nucleotide sequence in the sense ofthe present invention is understood as meaning an isolated and/orpurified nucleotide sequence having at least a percentage identity withthe bases of a nucleotide sequence according to the invention of atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7%, thispercentage being purely statistical and it being possible to distributethe differences between the two nucleotide sequences at random and overthe whole of their length.

Specific homologous nucleotide sequence in the sense of the presentinvention is understood as meaning a homologous nucleotide sequencehaving at least one nucleotide sequence of a specific fragment, such asdefined above. The “specific” homologous sequences can comprise, forexample, the sequences corresponding to the genomic sequence or to thesequences of its fragments representative of variants of the genome ofAlicyclobacillus acidocaldarius. These specific homologous sequences canthus correspond to variations linked to mutations within strains ofAlicyclobacillus acidocaldarius, and especially correspond totruncations, substitutions, deletions and/or additions of at least onenucleotide. The homologous sequences can likewise correspond tovariations linked to the degeneracy of the genetic code.

The term “degree or percentage of sequence homology” refers to “degreeor percentage of sequence identity between two sequences after optimalalignment” as defined in the present application.

Two amino-acids or nucleotidic sequences are said to be “identical” ifthe sequence of amino-acids or nucleotidic residues, in the twosequences is the same when aligned for maximum correspondence asdescribed below. Sequence comparisons between two (or more) peptides orpolynucleotides are typically performed by comparing sequences of twooptimally aligned sequences over a segment or “comparison window” toidentify and compare local regions of sequence similarity. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman, Ad. App. Math 2:482 (1981), bythe homology alignment algorithm of Neddleman and Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerizedimplementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575 Science Dr., Madison, Wis.), or by visual inspection.

“Percentage of sequence identity” (or degree of identity) is determinedby comparing two optimally aligned sequences over a comparison window,where the portion of the peptide or polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalamino-acid residue or nucleic acid base occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

The definition of sequence identity given above is the definition thatwould be used by one of skill in the art. The definition by itself doesnot need the help of any algorithm, the algorithms being helpful only toachieve the optimal alignments of sequences, rather than the calculationof sequence identity.

From the definition given above, it follows that there is a well-definedand only one value for the sequence identity between two comparedsequences, which value corresponds to the value obtained for the best oroptimal alignment.

In the BLAST N or BLAST P “BLAST 2 sequence,” software, which isavailable at the website ncbi.nlm.nih.gov/gorf/bl2.html, and habituallyused by the inventors and in general by the skilled person for comparingand determining the identity between two sequences, gap cost, whichdepends on the sequence length to be compared, is directly selected bythe software (i.e., 11.2 for substitution matrix BLOSUM-62 forlength>85).

Complementary nucleotide sequence of a sequence of the invention isunderstood as meaning any DNA whose nucleotides are complementary tothose of the sequence of the invention, and whose orientation isreversed (antisense sequence).

Hybridization under conditions of stringency with a nucleotide sequenceaccording to the invention is understood as meaning hybridization underconditions of temperature and ionic strength chosen in such a way thatthey allow the maintenance of the hybridization between two fragments ofcomplementary DNA.

By way of illustration, conditions of great stringency of thehybridization step with the aim of defining the nucleotide fragmentsdescribed above are advantageously the following.

The hybridization is carried out at a preferential temperature of 65° C.in the presence of SSC buffer, 1×SSC corresponding to 0.15 M NaCl and0.05 M Na citrate. The washing steps, for example, can be the following:2×SSC, at ambient temperature followed by two washes with 2×SSC, 0.5%SDS at 65° C.; 2×0.5×SSC, 0.5% SDS; at 65° C. for 10 minutes each.

The conditions of intermediate stringency, using, for example, atemperature of 42° C. in the presence of a 2×SSC buffer, or of lessstringency, for example, a temperature of 37° C. in the presence of a2×SSC buffer, respectively, require a globally less significantcomplementarity for the hybridization between the two sequences.

The stringent hybridization conditions described above for apolynucleotide with a size of approximately 350 bases will be adapted bya person skilled in the art for oligonucleotides of greater or smallersize, according to the teachings of Sambrook et al., 1989.

Among the isolated and/or purified nucleotide sequences according to theinvention, are those that can be used as a primer or probe in methodsallowing the homologous sequences according to the invention to beobtained, these methods, such as the polymerase chain reaction (PCR),nucleic acid cloning, and sequencing, being well known to a personskilled in the art.

Among the isolated and/or purified nucleotide sequences according to theinvention, those are again preferred that can be used as a primer orprobe in methods allowing the presence of SEQ ID NOS:2, 19, 36, 53, 70,87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274, 291, 308,325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495, 512, 529, 546,563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733, 750, 767, 784,801, 818, 835, 852, 869, 886, 903, 920, 937, 954, 971, 988, 1005, 1022,1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158, 1175, 1192, 1209, 1226,1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362, 1379, 1396, 1413, 1430,1447, 1464, 1481, 1498, 1515, 1532, 1549, and 1566, one of theirfragments, or one of their variants such as defined below to bediagnosed.

The nucleotide sequence fragments according to the invention can beobtained, for example, by specific amplification, such as PCR, or afterdigestion with appropriate restriction enzymes of nucleotide sequencesaccording to the invention, these methods in particular being describedin the work of Sambrook et al., 1989. Such representative fragments canlikewise be obtained by chemical synthesis according to methods wellknown to persons of ordinary skill in the art.

Modified nucleotide sequence will be understood as meaning anynucleotide sequence obtained by mutagenesis according to techniques wellknown to a person skilled in the art, and containing modifications withrespect to the normal sequences according to the invention, for example,mutations in the regulatory and/or promoter sequences of polypeptideexpression, especially leading to a modification of the rate ofexpression of the polypeptide or to a modulation of the replicativecycle.

Modified nucleotide sequence will likewise be understood as meaning anynucleotide sequence coding for a modified polypeptide, such as definedbelow.

The present invention relates to nucleotide sequence comprising isolatedand/or purified nucleotide sequences of Alicyclobacillus acidocaldarius,characterized in that they are selected from the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954,971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158,1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362,1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and1566 or one of their fragments.

Embodiments of the invention likewise relate to isolated and/or purifiednucleotide sequences characterized in that they comprise a nucleotidesequence selected from: a) at least one of a nucleotide sequence of SEQID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223,240, 257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461,478, 495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699,716, 733, 750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937,954, 971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141,1158, 1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345,1362, 1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549,and 1566 or one of their fragments or one of their fragments; b) anucleotide sequence of a specific fragment of a sequence such as definedin a); c) a homologous nucleotide sequence having at least 80% identitywith a sequence such as defined in a) or b); d) a complementarynucleotide sequence or sequence of RNA corresponding to a sequence suchas defined in a), b) or c); and e) a nucleotide sequence modified by asequence such as defined in a), b), c) or d).

Among the isolated and/or purified nucleotide sequences according to theinvention are the nucleotide sequences of SEQ ID NOS:13-17, 30-34,47-51, 64-68, 81-85, 98-102, 115-119, 132-136, 149-153, 166-170,183-187, 200-204, 217-221, 234-238, 251-255, 268-272, 285-289, 302-306,319-323, 336-340, 353-357, 370-374, 387-391, 404-408, 421-425, 438-442,455-459, 472-476, 489-493, 506-510, 523-527, 540-544, 557-561, 574-578,591-595, 608-612, 625-629, 642-646, 659-663, 676-680, 693-697, 710-714,727-731, 744-748, 761-765, 778-782, 795-799, 812-816, 829-833, 846-850,863-867, 880-884, 897-901, 914-918, 931-935, 948-952, 965-969, 982-986,999-1003, 1016-1020, 1033-1037, 1050-1054, 1067-1071, 1084-1088,1101-1105, 1118-1122, 1135-1139, 1152-1156, 1169-1173, 1186-1190,1203-1207, 1220-1224, 1237-1241, 1254-1258, 1271-1275, 1288-1292,1305-1309, 1322-1326, 1339-1343, 1356-1360, 1373-1377, 1390-1394,1407-1411, 1424-1428, 1441-1445, 1458-1462, 1475-1479, 1492-1496,1509-1513, 1526-1530, 1543-1547, 1560-1564, and 1577-1581, or fragmentsthereof and any isolated and/or purified nucleotide sequences, whichhave a homology of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or99.7% identity with the at least one of the sequences of SEQ ID NOS:2,19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257,274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495,512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733,750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954, 971,988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158, 1175,1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362, 1379,1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and 1566 orfragments thereof. Such homologous sequences can comprise, for example,the sequences corresponding to the genomic sequences of Alicyclobacillusacidocaldarius. In the same manner, these specific homologous sequencescan correspond to variations linked to mutations within strains ofAlicyclobacillus acidocaldarius and especially correspond totruncations, substitutions, deletions and/or additions of at least onenucleotide. As will be apparent to one of ordinary skill in the art,such homologues are easily created and identified using conventionaltechniques and publicly available computer programs such as BLAST.Accordingly, each homologue referenced above should be considered as setforth herein and fully described.

Embodiments of the invention comprise the isolated and/or purifiedpolypeptides coded for by a nucleotide sequence according to theinvention, or fragments thereof, whose sequence is represented by afragment. Amino acid sequences corresponding to the isolated and/orpurified polypeptides that can be coded for according to one of thethree possible reading frames of at least one of the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954,971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158,1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362,1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and1566.

Embodiments of the invention likewise relate to the isolated and/orpurified polypeptides, characterized in that they comprise a polypeptideselected from at least one of the amino acid sequences of SEQ ID NOS:1,18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256,273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494,511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732,749, 766, 783, 800, 817, 834, 851, 868, 885, 902, 819, 936, 953, 970,987, 1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123, 1140, 1157, 1174,1191, 1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327, 1344, 1361, 1378,1395, 1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531, 1548, and 1565 orone of their fragments.

Among the isolated and/or purified polypeptides, according toembodiments of the invention, are the isolated and/or purifiedpolypeptides of amino acid sequence SEQ ID NOS:8-12, 25-29, 42-46,59-63, 76-80, 93-97, 110-114, 127-131, 144-148, 161-165, 178-182,195-199, 212-216, 229-233, 246-250, 263-267, 280-284, 297-301, 314-318,331-335, 348-352, 365-369, 382-386, 399-403, 416-420, 433-437, 450-454,467-471, 484-488, 501-505, 518-522, 535-539, 552-556, 569-573, 586-590,603-607, 620-624, 637-641, 654-658, 671-675, 688-692, 705-709, 722-726,739-743, 756-760, 773-777, 790-794, 807-811, 824-828, 841-845, 858-862,875-879, 892-896, 909-913, 926-930, 943-947, 960-964, 977-981, 994-998,1011-1015, 1028-1032, 1045-1049, 1062-1066, 1079-1083, 1096-1100,1113-1117, 1130-1134, 1147-1151, 1164-1168, 1181-1185, 1198-1202,1215-1219, 1232-1236, 1249-1253, 1266-1270, 1283-1287, 1300-1304,1317-1321, 1334-1338, 1351-1355, 1368-1372, 1385-1389, 1402-1406,1419-1423, 1436-1440, 1453-1457, 1470-1474, 1487-1491, 1504-1508,1521-1525, 1538-1542, 1555-1559, and 1572-1576, or fragments thereof orany other isolated and/or purified polypeptides that have a homology ofat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7% identitywith at least one of the sequences of SEQ ID NOS:1, 18, 35, 52, 69, 86,103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 324,341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545, 562,579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, 766, 783, 800,817, 834, 851, 868, 885, 902, 819, 936, 953, 970, 987, 1004, 1021, 1038,1055, 1072, 1089, 1106, 1123, 1140, 1157, 1174, 1191, 1208, 1225, 1242,1259, 1276, 1293, 1310, 1327, 1344, 1361, 1378, 1395, 1412, 1429, 1446,1463, 1480, 1497, 1514, 1531, 1548, and 1565 or fragments thereof. Aswill be apparent to one of ordinary skill in the art, such homologuesare easily created and identified using conventional techniques andpublicly available computer programs such as BLAST. Accordingly, eachhomologue referenced above should be considered as set forth herein andfully described.

Embodiments of the invention also relate to the polypeptides,characterized in that they comprise a polypeptide selected from: a) aspecific fragment of at least 5 amino acids of a polypeptide of an aminoacid sequence according to the invention; b) a polypeptide homologous toa polypeptide such as defined in a); c) a specific biologically activefragment of a polypeptide such as defined in a) or b); and d) apolypeptide modified by a polypeptide such as defined in a), b) or c).

In the present description, the terms polypeptide, peptide and proteinare interchangeable.

In embodiments of the invention, the isolated and/or purifiedpolypeptides according to the invention may be glycosylated, pegylated,and/or otherwise post-translationally modified. In further embodiments,glycosylation, pegylation, and/or other post-translational modificationsmay occur in vivo or in vitro and/or may be performed using chemicaltechniques. In additional embodiments, any glycosylation, pegylationand/or other post-translational modifications may be N-linked orO-linked.

In embodiments of the invention any one of the isolated and/or purifiedpolypeptides according to the invention may be enzymatically orfunctionally active at temperatures at or above about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/ormay be enzymatically or functionally active at a pH at, below, and/orabove 8, 7, 6, 5, 4, 3, 2, 1, and/or 0. In further embodiments of theinvention, glycosylation, pegylation, and/or other post-translationalmodification may be required for the isolated and/or purifiedpolypeptides according to the invention to be enzymatically orfunctionally active at a pH at or below 8, 7, 6, 5, 4, 3, 2, 1, and/or 0or at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, and/or 95 degrees Celsius.

Aspects of the invention relate to polypeptides that are isolated orobtained by purification from natural sources, or else obtained bygenetic recombination, or alternatively by chemical synthesis and thatthey may thus contain unnatural amino acids, as will be described below.

A “polypeptide fragment” according to the embodiments of the inventionis understood as designating a polypeptide containing at least 5consecutive amino acids, preferably 10 consecutive amino acids or 15consecutive amino acids.

In the present invention, a specific polypeptide fragment is understoodas designating the consecutive polypeptide fragment coded for by aspecific fragment nucleotide sequence according to the invention.

“Homologous polypeptide” will be understood as designating thepolypeptides having, with respect to the natural polypeptide, certainmodifications such as, in particular, a deletion, addition, orsubstitution of at least one amino acid, a truncation, a prolongation, achimeric fusion, and/or a mutation. Among the homologous polypeptides,those are preferred whose amino acid sequence has at least 80% or 90%,homology with the sequences of amino acids of polypeptides according tothe invention.

“Specific homologous polypeptide” will be understood as designating thehomologous polypeptides such as defined above and having a specificfragment of polypeptide according to the invention.

In the case of a substitution, one or more consecutive or nonconsecutiveamino acids are replaced by “equivalent” amino acids. The expression“equivalent” amino acid is directed here at designating any amino acidcapable of being substituted by one of the amino acids of the basestructure without, however, essentially modifying the biologicalactivities of the corresponding peptides and such that they will bedefined by the following. As will be apparent to one of ordinary skillin the art, such substitutions are easily created and identified usingstandard molecular biology techniques and publicly available computerprograms such as BLAST. Accordingly, each substitution referenced aboveshould be considered as set forth herein and fully described. Examplesof such substitutions in the amino acid sequences of SEQ ID NOS:1, 18,35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273,290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511,528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749,766, 783, 800, 817, 834, 851, 868, 885, 902, 819, 936, 953, 970, 987,1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123, 1140, 1157, 1174, 1191,1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327, 1344, 1361, 1378, 1395,1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531, 1548, and 1565 mayinclude those isolated and/or purified polypeptides of amino acidsequence SEQ ID NOS:8-12, 25-29, 42-46, 59-63, 76-80, 93-97, 110-114,127-131, 144-148, 161-165, 178-182, 195-199, 212-216, 229-233, 246-250,263-267, 280-284, 297-301, 314-318, 331-335, 348-352, 365-369, 382-386,399-403, 416-420, 433-437, 450-454, 467-471, 484-488, 501-505, 518-522,535-539, 552-556, 569-573, 586-590, 603-607, 620-624, 637-641, 654-658,671-675, 688-692, 705-709, 722-726, 739-743, 756-760, 773-777, 790-794,807-811, 824-828, 841-845, 858-862, 875-879, 892-896, 909-913, 926-930,943-947, 960-964, 977-981, 994-998, 1011-1015, 1028-1032, 1045-1049,1062-1066, 1079-1083, 1096-1100, 1113-1117, 1130-1134, 1147-1151,1164-1168, 1181-1185, 1198-1202, 1215-1219, 1232-1236, 1249-1253,1266-1270, 1283-1287, 1300-1304, 1317-1321, 1334-1338, 1351-1355,1368-1372, 1385-1389, 1402-1406, 1419-1423, 1436-1440, 1453-1457,1470-1474, 1487-1491, 1504-1508, 1521-1525, 1538-1542, 1555-1559, and1572-1576. These equivalent amino acids may be determined either bydepending on their structural homology with the amino acids that theysubstitute, or on results of comparative tests of biological activitybetween the different polypeptides, which are capable of being carriedout.

By way of non-limiting example, the possibilities of substitutionscapable of being carried out without resulting in an extensivemodification of the biological activity of the corresponding modifiedpolypeptides will be mentioned, the replacement, for example, of leucineby valine or isoleucine, of aspartic acid by glutamic acid, of glutamineby asparagine, of arginine by lysine, etc., the reverse substitutionsnaturally being envisageable under the same conditions.

In a further embodiment, substitutions are limited to substitutions inamino acids not conserved among other proteins that have similaridentified enzymatic activity. For example, one of ordinary skill in theart may align proteins of the same function in similar organisms anddetermine which amino acids are generally conserved among proteins ofthat function. One example of a program that may be used to generatesuch alignments is available at the web site charite.de/bioinf/strap/ inconjunction with the databases provided by the NCBI. Examples of suchpolypeptides may include, but are not limited to, those found in aminoacid sequence SEQ ID NOS:8-12, 25-29, 42-46, 59-63, 76-80, 93-97,110-114, 127-131, 144-148, 161-165, 178-182, 195-199, 212-216, 229-233,246-250, 263-267, 280-284, 297-301, 314-318, 331-335, 348-352, 365-369,382-386, 399-403, 416-420, 433-437, 450-454, 467-471, 484-488, 501-505,518-522, 535-539, 552-556, 569-573, 586-590, 603-607, 620-624, 637-641,654-658, 671-675, 688-692, 705-709, 722-726, 739-743, 756-760, 773-777,790-794, 807-811, 824-828, 841-845, 858-862, 875-879, 892-896, 909-913,926-930, 943-947, 960-964, 977-981, 994-998, 1011-1015, 1028-1032,1045-1049, 1062-1066, 1079-1083, 1096-1100, 1113-1117, 1130-1134,1147-1151, 1164-1168, 1181-1185, 1198-1202, 1215-1219, 1232-1236,1249-1253, 1266-1270, 1283-1287, 1300-1304, 1317-1321, 1334-1338,1351-1355, 1368-1372, 1385-1389, 1402-1406, 1419-1423, 1436-1440,1453-1457, 1470-1474, 1487-1491, 1504-1508, 1521-1525, 1538-1542,1555-1559, and 1572-1576.

Thus, according to one embodiment of the invention, substitutions ormutations may be made at positions that are generally conserved amongproteins of that function. In a further embodiment, nucleic acidsequences may be mutated or substituted such that the amino acid theycode for is unchanged (degenerate substitutions and/or mutations) and/ormutated or substituted such that any resulting amino acid substitutionsor mutations are made at positions that are generally conserved amongproteins of that function. Examples of such nucleic acid sequences mayinclude, but are not limited to, those found in are the nucleotidesequences of SEQ ID NOS:13-17, 30-34, 47-51, 64-68, 81-85, 98-102,115-119, 132-136, 149-153, 166-170, 183-187, 200-204, 217-221, 234-238,251-255, 268-272, 285-289, 302-306, 319-323, 336-340, 353-357, 370-374,387-391, 404-408, 421-425, 438-442, 455-459, 472-476, 489-493, 506-510,523-527, 540-544, 557-561, 574-578, 591-595, 608-612, 625-629, 642-646,659-663, 676-680, 693-697, 710-714, 727-731, 744-748, 761-765, 778-782,795-799, 812-816, 829-833, 846-850, 863-867, 880-884, 897-901, 914-918,931-935, 948-952, 965-969, 982-986, 999-1003, 1016-1020, 1033-1037,1050-1054, 1067-1071, 1084-1088, 1101-1105, 1118-1122, 1135-1139,1152-1156, 1169-1173, 1186-1190, 1203-1207, 1220-1224, 1237-1241,1254-1258, 1271-1275, 1288-1292, 1305-1309, 1322-1326, 1339-1343,1356-1360, 1373-1377, 1390-1394, 1407-1411, 1424-1428, 1441-1445,1458-1462, 1475-1479, 1492-1496, 1509-1513, 1526-1530, 1543-1547,1560-1564, 1577-1581 or fragments thereof.

The specific homologous polypeptides likewise correspond to polypeptidescoded for by the specific homologous nucleotide sequences such asdefined above and thus comprise in the present definition thepolypeptides, which are mutated or correspond to variants that can existin Alicyclobacillus acidocaldarius, and that especially correspond totruncations, substitutions, deletions, and/or additions of at least oneamino acid residue.

“Specific biologically active fragment of a polypeptide” according to anembodiment of the invention will be understood in particular asdesignating a specific polypeptide fragment, such as defined above,having at least one of the characteristics of polypeptides according tothe invention. In certain embodiments the peptide is capable of behavingas at least one of the types of proteins outlined in Table 1.

The polypeptide fragments according to embodiments of the invention cancorrespond to isolated or purified fragments naturally present inAlicyclobacillus acidocaldarius or correspond to fragments that can beobtained by cleavage of the polypeptide by a proteolytic enzyme, such astrypsin or chymotrypsin or collagenase, or by a chemical reagent, suchas cyanogen bromide (CNBr). Such polypeptide fragments can likewise justas easily be prepared by chemical synthesis, from hosts transformed byan expression vector according to the invention containing a nucleicacid allowing the expression of the fragments, placed under the controlof appropriate regulation and/or expression elements.

“Modified polypeptide” of a polypeptide according to an embodiment ofthe invention is understood as designating a polypeptide obtained bygenetic recombination or by chemical synthesis as will be describedbelow, having at least one modification with respect to the normalsequence. These modifications may or may not be able to bear on aminoacids at the origin of specificity, and/or of activity, or at the originof the structural conformation, localization, and of the capacity ofmembrane insertion of the polypeptide according to the invention. Itwill thus be possible to create polypeptides of equivalent, increased,or decreased activity, and of equivalent, narrower, or widerspecificity. Among the modified polypeptides, it is necessary to mentionthe polypeptides in which up to 5 or more amino acids can be modified,truncated at the N- or C-terminal end, or even deleted or added.

The methods allowing the modulations on eukaryotic or prokaryotic cellsto be demonstrated are well known to a person of ordinary skill in theart. It is likewise well understood that it will be possible to use thenucleotide sequences coding for the modified polypeptides for themodulations, for example, through vectors according to the invention anddescribed below.

The preceding modified polypeptides can be obtained by usingcombinatorial chemistry, in which it is possible to systematically varyparts of the polypeptide before testing them on models, cell cultures ormicroorganisms, for example, to select the compounds that are mostactive or have the properties sought.

Chemical synthesis likewise has the advantage of being able to usenonnatural amino acids, or nonpeptide bonds.

Thus, in order to improve the duration of life of the polypeptidesaccording to the invention, it may be of interest to use nonnaturalamino acids, for example, in D form, or else amino acid analogs,especially sulfur-containing forms, for example.

Finally, it will be possible to integrate the structure of thepolypeptides according to the invention, its specific or modifiedhomologous forms, into chemical structures of polypeptide type orothers. Thus, it may be of interest to provide at the N- and C-terminalends molecules not recognized by proteases.

The nucleotide sequences coding for a polypeptide according to theinvention are likewise part of the invention.

The invention likewise relates to nucleotide sequences utilizable as aprimer or probe, characterized in that the sequences are selected fromthe nucleotide sequences according to the invention.

It is well understood that the present invention, in variousembodiments, likewise relates to specific polypeptides ofAlicyclobacillus acidocaldarius, coded for by nucleotide sequences,capable of being obtained by purification from natural polypeptides, bygenetic recombination or by chemical synthesis by procedures well knownto a person skilled in the art and such as described in particularbelow. In the same manner, the labeled or unlabeled mono- or polyclonalantibodies directed against the specific polypeptides coded for by thenucleotide sequences are also encompassed by the invention.

Embodiments of the invention additionally relate to the use of anucleotide sequence according to the invention as a primer or probe forthe detection and/or the amplification of nucleic acid sequences.

The nucleotide sequences according to embodiments of the invention canthus be used to amplify nucleotide sequences, especially by the PCRtechnique (polymerase chain reaction) (Erlich, 1989; Innis et al., 1990;Rolfs et al., 1991; and White et al., 1997).

These oligodeoxyribonucleotide or oligoribonucleotide primersadvantageously have a length of at least 8 nucleotides, preferably of atleast 12 nucleotides, and even more preferentially of at least 20nucleotides.

Other amplification techniques of the target nucleic acid can beadvantageously employed as alternatives to PCR.

The nucleotide sequences of the invention, in particular the primersaccording to the invention, can likewise be employed in other proceduresof amplification of a target nucleic acid, such as: the TAS technique(Transcription-based Amplification System), described by Kwoh et al. in1989; the 3SR technique (Self-Sustained Sequence Replication), describedby Guatelli et al. in 1990; the NASBA technique (Nucleic Acid SequenceBased Amplification), described by Kievitis et al. in 1991; the SDAtechnique (Strand Displacement Amplification) (Walker et al., 1992); andthe TMA technique (Transcription Mediated Amplification).

The polynucleotides of the invention can also be employed in techniquesof amplification or of modification of the nucleic acid serving as aprobe, such as: the LCR technique (Ligase Chain Reaction), described byLandegren et al. in 1988 and improved by Barany et al. in 1991, whichemploys a thermostable ligase; the RCR technique (Repair ChainReaction), described by Segev in 1992; the CPR technique (Cycling ProbeReaction), described by Duck et al. in 1990; the amplification techniquewith Q-beta replicase, described by Miele et al. in 1983 and especiallyimproved by Chu et al. in 1986, Lizardi et al. in 1988, then by Burg etal., as well as by Stone et al. in 1996.

In the case where the target polynucleotide to be detected is possiblyan RNA, for example, an mRNA, it will be possible to use, prior to theemployment of an amplification reaction with the aid of at least oneprimer according to the invention or to the employment of a detectionprocedure with the aid of at least one probe of the invention, an enzymeof reverse transcriptase type in order to obtain a cDNA from the RNAcontained in the biological sample. The cDNA obtained will thus serve asa target for the primer(s) or the probe(s) employed in the amplificationor detection procedure according to the invention.

The detection probe will be chosen in such a manner that it hybridizeswith the target sequence or the amplicon generated from the targetsequence. By way of sequence, such a probe will advantageously have asequence of at least 12 nucleotides, in particular of at least 20nucleotides, and preferably of at least 100 nucleotides.

Embodiments of the invention also comprise the nucleotide sequencesutilizable as a probe or primer according to the invention,characterized in that they are labeled with a radioactive compound orwith a nonradioactive compound.

The unlabeled nucleotide sequences can be used directly as probes orprimers, although the sequences are generally labeled with a radioactiveisotope (³²P, ³⁵S, ³H, ¹²⁵I) or with a nonradioactive molecule (biotin,acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine, fluorescein) toobtain probes that are utilizable for numerous applications.

Examples of nonradioactive labeling of nucleotide sequences aredescribed, for example, in French Patent No. 7810975 or by Urdea et al.or by Sanchez-Pescador et al. in 1988.

In the latter case, it will also be possible to use one of the labelingmethods described in patents FR-2 422 956 and FR-2 518 755.

The hybridization technique can be carried out in various manners(Matthews et al., 1988). The most general method consists inimmobilizing the nucleic acid extract of cells on a support (such asnitrocellulose, nylon, polystyrene) and in incubating, underwell-defined conditions, the immobilized target nucleic acid with theprobe. After hybridization, the excess of probe is eliminated and thehybrid molecules formed are detected by the appropriate method(measurement of the radioactivity, of the fluorescence or of theenzymatic activity linked to the probe).

The invention, in various embodiments, likewise comprises the nucleotidesequences according to the invention, characterized in that they areimmobilized on a support, covalently or noncovalently.

According to another advantageous mode of employing nucleotide sequencesaccording to the invention, the latter can be used immobilized on asupport and can thus serve to capture, by specific hybridization, thetarget nucleic acid obtained from the biological sample to be tested. Ifnecessary, the solid support is separated from the sample and thehybridization complex formed between the capture probe and the targetnucleic acid is then detected with the aid of a second probe, aso-called detection probe, labeled with an easily detectable element.

Another aspect of the present invention is a vector for the cloningand/or expression of a sequence, characterized in that it contains anucleotide sequence according to the invention.

The vectors, according to the invention, characterized in that theycontain the elements allowing the integration, expression and/or thesecretion of the nucleotide sequences in a determined host cell, arelikewise part of the invention.

The vector may then contain a promoter, signals of initiation andtermination of translation, as well as appropriate regions of regulationof transcription. It may be able to be maintained stably in the hostcell and can optionally have particular signals specifying the secretionof the translated protein. These different elements may be chosen as afunction of the host cell used. To this end, the nucleotide sequencesaccording to the invention may be inserted into autonomous replicationvectors within the chosen host, or integrated vectors of the chosenhost.

Such vectors will be prepared according to the methods currently used bya person skilled in the art, and it will be possible to introduce theclones resulting therefrom into an appropriate host by standard methods,such as, for example, lipofection, electroporation, and thermal shock.

The vectors according to the invention are, for example, vectors ofplasmid or viral origin. One example of a vector for the expression ofpolypeptides of the invention is Baculovirus.

These vectors are useful for transforming host cells in order to cloneor to express the nucleotide sequences of the invention.

The invention likewise comprises the host cells transformed by a vectoraccording to the invention.

These cells can be obtained by the introduction into host cells of anucleotide sequence inserted into a vector such as defined above, thenthe culturing of the cells under conditions allowing the replicationand/or expression of the transfected nucleotide sequence.

The host cell can be selected from prokaryotic or eukaryotic systems,such as, for example, bacterial cells (Olins and Lee, 1993), butlikewise yeast cells (Buckholz, 1993), as well as plants cells, such asArabidopsis sp., and animal cells, in particular the cultures ofmammalian cells (Edwards and Aruffo, 1993), for example, Chinese hamsterovary (CHO) cells, but likewise the cells of insects in which it ispossible to use procedures employing baculoviruses, for example, 519insect cells (Luckow, 1993).

Embodiments of the invention likewise relate to organisms comprising oneof the transformed cells according to the invention.

The obtainment of transgenic organisms, according to the invention, ofexpressing one or more of the genes of Alicyclobacillus acidocaldariusor part of the genes may be carried out in, for example, rats, mice, orrabbits according to methods well known to a person skilled in the art,such as by viral or nonviral transfections. It will be possible toobtain the transgenic organisms expressing one or more of the genes bytransfection of multiple copies of the genes under the control of astrong promoter of ubiquitous nature, or selective for one type oftissue. It will likewise be possible to obtain the transgenic organismsby homologous recombination in embryonic cell strains, transfer of thesecell strains to embryos, selection of the affected chimeras at the levelof the reproductive lines, and growth of the chimeras.

The transformed cells, as well as the transgenic organisms according tothe invention, are utilizable in procedures for preparation ofrecombinant polypeptides.

It is today possible to produce recombinant polypeptides in relativelylarge quantity by genetic engineering using the cells transformed byexpression vectors according to the invention or using transgenicorganisms according to the invention.

The procedures for preparation of a polypeptide of the invention inrecombinant form, characterized in that they employ a vector and/or acell transformed by a vector according to the invention and/or atransgenic organism comprising one of the transformed cells according tothe invention are themselves comprised in the present invention.

As used herein, “transformation” and “transformed” relate to theintroduction of nucleic acids into a cell, whether prokaryotic oreukaryotic. Further, “transformation” and “transformed,” as used herein,need not relate to growth control or growth deregulation.

Among the procedures for preparation of a polypeptide of the inventionin recombinant form, the preparation procedures employing a vector,and/or a cell transformed by the vector and/or a transgenic organismcomprising one of the transformed cells, containing a nucleotidesequence according to the invention coding for a polypeptide ofAlicyclobacillus acidocaldarius.

A variant according to the invention may consist of producing arecombinant polypeptide fused to a “carrier” protein (chimeric protein).The advantage of this system is that it may allow stabilization ofand/or a decrease in the proteolysis of the recombinant product, anincrease in the solubility in the course of renaturation in vitro and/ora simplification of the purification when the fusion partner has anaffinity for a specific ligand.

More particularly, the invention relates to a procedure for preparationof a polypeptide of the invention comprising the following steps: a)culture of transformed cells under conditions allowing the expression ofa recombinant polypeptide of a nucleotide sequence according to theinvention; b) if need be, recovery of the recombinant polypeptide.

When the procedure for preparation of a polypeptide of the inventionemploys a transgenic organism according to the invention, therecombinant polypeptide is then extracted from the organism.

The invention also relates to a polypeptide that is capable of beingobtained by a procedure of the invention such as described previously.

The invention also comprises a procedure for preparation of a syntheticpolypeptide, characterized in that it uses a sequence of amino acids ofpolypeptides according to the invention.

The invention likewise relates to a synthetic polypeptide obtained by aprocedure according to the invention.

The polypeptides according to the invention can likewise be prepared bytechniques that are conventional in the field of the synthesis ofpeptides. This synthesis can be carried out in homogeneous solution orin solid phase.

For example, recourse can be made to the technique of synthesis in anhomogeneous solution described by Houben-Weyl in 1974.

This method of synthesis consists in successively condensing, two bytwo, the successive amino acids in the order required, or in condensingamino acids and fragments formed previously and already containingseveral amino acids in the appropriate order, or alternatively severalfragments previously prepared in this way, it being understood that itwill be necessary to protect beforehand all the reactive functionscarried by these amino acids or fragments, with the exception of aminefunctions of one and carboxyls of the other or vice-versa, which mustnormally be involved in the formation of peptide bonds, especially afteractivation of the carboxyl function, according to the methods well knownin the synthesis of peptides.

Recourse may also be made to the technique described by Merrifield.

To make a peptide chain according to the Merrifield procedure, recourseis made to a very porous polymeric resin, on which is immobilized thefirst C-terminal amino acid of the chain. This amino acid is immobilizedon a resin through its carboxyl group and its amine function isprotected. The amino acids that are going to form the peptide chain arethus immobilized, one after the other, on the amino group, which isdeprotected beforehand each time, of the portion of the peptide chainalready formed, and which is attached to the resin. When the whole ofthe desired peptide chain has been formed, the protective groups of thedifferent amino acids forming the peptide chain are eliminated and thepeptide is detached from the resin with the aid of an acid.

The invention additionally relates to hybrid polypeptides having atleast one polypeptide according to the invention, and a sequence of apolypeptide capable of inducing an immune response in man or animals.

Advantageously, the antigenic determinant is such that it is capable ofinducing a humoral and/or cellular response.

It will be possible for such a determinant to comprise a polypeptideaccording to the invention in glycosylated, pegylated, and/or otherwisepost-translationally modified form used with a view to obtainingimmunogenic compositions capable of inducing the synthesis of antibodiesdirected against multiple epitopes.

These hybrid molecules can be formed, in part, of a polypeptide carriermolecule or of fragments thereof according to the invention, associatedwith a possibly immunogenic part, in particular, an epitope of thediphtheria toxin, the tetanus toxin, a surface antigen of the hepatitisB virus (Patent FR 79 21811), the VP1 antigen of the poliomyelitis virusor any other viral or bacterial toxin or antigen.

The procedures for synthesis of hybrid molecules encompass the methodsused in genetic engineering for constructing hybrid nucleotide sequencescoding for the polypeptide sequences sought. It will be possible, forexample, to refer advantageously to the technique for obtainment ofgenes coding for fusion proteins described by Minton in 1984.

The hybrid nucleotide sequences coding for a hybrid polypeptide as wellas the hybrid polypeptides according to the invention characterized inthat they are recombinant polypeptides obtained by the expression of thehybrid nucleotide sequences are likewise part of the invention.

The invention likewise comprises the vectors characterized in that theycontain one of the hybrid nucleotide sequences. The host cellstransformed by the vectors, the transgenic organisms comprising one ofthe transformed cells as well as the procedures for preparation ofrecombinant polypeptides using the vectors, the transformed cells and/orthe transgenic organisms are, of course, likewise part of the invention.

The polypeptides according to the invention, the antibodies according tothe invention described below and the nucleotide sequences according tothe invention can advantageously be employed in procedures for thedetection and/or identification of Alicyclobacillus acidocaldarius, in asample capable of containing them. These procedures, according to thespecificity of the polypeptides, the antibodies and the nucleotidesequences according to the invention that will be used, will inparticular be able to detect and/or to identify Alicyclobacillusacidocaldarius.

The polypeptides according to the invention can advantageously beemployed in a procedure for the detection and/or the identification ofAlicyclobacillus acidocaldarius in a sample capable of containing them,characterized in that it comprises the following steps: a) contacting ofthis sample with a polypeptide or one of its fragments according to theinvention (under conditions allowing an immunological reaction betweenthe polypeptide and the antibodies possibly present in the biologicalsample); and b) demonstration of the antigen-antibody complexes possiblyformed.

Any conventional procedure can be employed for carrying out such adetection of the antigen-antibody complexes possibly formed.

By way of non-limiting example, one method brings into playimmunoenzymatic processes according to the ELISA technique, byimmunofluorescence, or radioimmunological processes (RIA) or theirequivalent.

Thus, the invention likewise relates to the polypeptides according tothe invention, labeled with the aid of an adequate label, such as, ofthe enzymatic, fluorescent or radioactive type.

Such methods comprise, for example, the following acts: deposition ofdetermined quantities of a polypeptide composition according to theinvention in the wells of a microtiter plate, introduction into thewells of increasing dilutions of serum, or of a biological sample otherthan that defined previously, having to be analyzed, incubation of thewells of the microtiter plate, introduction into the wells of themicrotiter plate of labeled antibodies directed against pigimmunoglobulins, the labeling of these antibodies having been carriedout with the aid of an enzyme selected from those that are capable ofhydrolyzing a substrate by modifying the absorption of the radiation ofthe latter, at least at a determined wavelength, for example at 550 nm,detection, by comparison with a control test, of the quantity ofhydrolyzed substrate.

The polypeptides according to the invention allow monoclonal orpolyclonal antibodies to be prepared, which are characterized in thatthey specifically recognize the polypeptides according to the invention.It will advantageously be possible to prepare the monoclonal antibodiesfrom hybridomas according to the technique described by Kohler andMilstein in 1975. It will be possible to prepare the polyclonalantibodies, for example, by immunization of an animal, in particular amouse, with a polypeptide or a DNA, according to the invention,associated with an adjuvant of the immune response, and thenpurification of the specific antibodies contained in the serum of theimmunized animals on an affinity column on which the polypeptide, whichhas served as an antigen, has previously been immobilized. Thepolyclonal antibodies according to the invention can also be prepared bypurification, on an affinity column on which a polypeptide according tothe invention has previously been immobilized, of the antibodiescontained in the serum of an animal immunologically challenged byAlicyclobacillus acidocaldarius, or a polypeptide or fragment accordingto the invention.

The invention likewise relates to mono- or polyclonal antibodies ortheir fragments, or chimeric antibodies, characterized in that they arecapable of specifically recognizing a polypeptide according to theinvention.

It will likewise be possible for the antibodies of the invention to belabeled in the same manner as described previously for the nucleicprobes of the invention, such as a labeling of enzymatic, fluorescent orradioactive type.

The invention is additionally directed at a procedure for the detectionand/or identification of Alicyclobacillus acidocaldarius in a sample,characterized in that it comprises the following steps: a) contacting ofthe sample with a mono- or polyclonal antibody according to theinvention (under conditions allowing an immunological reaction betweenthe antibodies and the polypeptides of Alicyclobacillus acidocaldariuspossibly present in the biological sample); and b) demonstration of theantigen-antibody complex possibly formed.

The present invention likewise relates to a procedure for the detectionand/or the identification of Alicyclobacillus acidocaldarius in asample, characterized in that it employs a nucleotide sequence accordingto the invention.

More particularly, the invention relates to a procedure for thedetection and/or the identification of Alicyclobacillus acidocaldariusin a sample, characterized in that it contains the following steps: a)if need be, isolation of the DNA from the sample to be analyzed; b)specific amplification of the DNA of the sample with the aid of at leastone primer, or a pair of primers, according to the invention; and c)demonstration of the amplification products.

These can be detected, for example, by the technique of molecularhybridization utilizing a nucleic probe according to the invention. Thisprobe will advantageously be labeled with a nonradioactive (cold probe)or radioactive isotope.

For the purposes of the present invention, “DNA of the biologicalsample” or “DNA contained in the biological sample” will be understoodas meaning either the DNA present in the biological sample considered,or possibly the cDNA obtained after the action of an enzyme of reversetranscriptase type on the RNA present in the biological sample.

A further embodiment of the invention comprises a method, characterizedin that it comprises the following acts: a) contacting of a nucleotideprobe according to the invention with a biological sample, the DNAcontained in the biological sample having, if need be, previously beenmade accessible to hybridization under conditions allowing thehybridization of the nucleotide probe with the DNA of the sample; and b)demonstration of the hybrid formed between the nucleotide probe and theDNA of the biological sample.

The present invention also relates to a procedure according to theinvention, characterized in that it comprises the following acts: a)contacting of a nucleotide probe immobilized on a support according tothe invention with a biological sample, the DNA of the sample having, ifneed be, previously been made accessible to hybridization, underconditions allowing the hybridization of the nucleotide probe with theDNA of the sample; b) contacting of the hybrid formed between thenucleotide probe immobilized on a support and the DNA contained in thebiological sample, if need be, after elimination of the DNA of thebiological sample that has not hybridized with the nucleotide probe,with a nucleotide probe labeled according to the invention; c)demonstration of the novel hybrid formed in act b).

According to an advantageous embodiment of the procedure for detectionand/or identification defined previously, this is characterized in that,prior to act a), the DNA of the biological sample is first amplifiedwith the aid of at least one primer according to the invention.

Embodiments of methods include methods of altering secondary metabolismin a cell, the methods comprising providing a recombinant, purified,and/or isolated nucleotide sequence comprising a nucleotide sequenceselected from the group consisting of a nucleotide sequence having atleast 90% sequence identity to at least one of the sequences of SEQ IDNOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240,257, 274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478,495, 512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716,733, 750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954,971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158,1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362,1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and1566 and/or a recombinant, purified, and/or isolated polypeptideselected from the group consisting of a polypeptide having at least 90%sequence identity to at least one of the sequences of SEQ ID NOS:1, 18,35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273,290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511,528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749,766, 783, 800, 817, 834, 851, 868, 885, 902, 819, 936, 953, 970, 987,1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123, 1140, 1157, 1174, 1191,1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327, 1344, 1361, 1378, 1395,1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531, 1548, and 1565 to thecell.

Further embodiments of methods include placing a cell producing orencoding a recombinant, purified, and/or isolated nucleotide sequencecomprising a nucleotide sequence selected from the group consisting of anucleotide sequence having at least 90% sequence identity to at leastone of the sequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138,155, 172, 189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376,393, 410, 427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597, 614,631, 648, 665, 682, 699, 716, 733, 750, 767, 784, 801, 818, 835, 852,869, 886, 903, 920, 937, 954, 971, 988, 1005, 1022, 1039, 1056, 1073,1090, 1107, 1124, 1141, 1158, 1175, 1192, 1209, 1226, 1243, 1260, 1277,1294, 1311, 1328, 1345, 1362, 1379, 1396, 1413, 1430, 1447, 1464, 1481,1498, 1515, 1532, 1549, and 1566 and/or a recombinant, purified, and/orisolated polypeptide selected from the group consisting of a polypeptidehaving at least 90% sequence identity to at least one of the sequencesof SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205,222, 239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443,460, 477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681,698, 715, 732, 749, 766, 783, 800, 817, 834, 851, 868, 885, 902, 819,936, 953, 970, 987, 1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123,1140, 1157, 1174, 1191, 1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327,1344, 1361, 1378, 1395, 1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531,1548, and 1565 in an environment comprising temperatures at or aboveabout 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95degrees Celsius and/or a pH at, below, and/or above 8, 7, 6, 5, 4, 3, 2,1, and/or 0.

The present invention provides cells that have been geneticallymanipulated to have an altered capacity to produce expressed proteins.In particular, the present invention relates to Gram-positivemicroorganisms, such as Bacillus species having enhanced expression of aprotein of interest, wherein one or more chromosomal genes have beeninactivated, and/or wherein one or more chromosomal genes have beendeleted from the Bacillus chromosome. In some further embodiments, oneor more indigenous chromosomal regions have been deleted from acorresponding wild-type Bacillus host chromosome. In furtherembodiments, the Bacillus is an Alicyclobacillus sp. or Alicyclobacillusacidocaldarius.

Additional embodiments, include methods of modulating metabolism attemperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, and/or 95 degrees Celsius and/or at a pH at, below,and/or above 8, 7, 6, 5, 4, 3, 2, 1, and/or 0 via providing arecombinant, purified, and/or isolated nucleotide sequence comprising anucleotide sequence selected from the group consisting of a nucleotidesequence having at least 90% sequence identity to at least one of thesequences of SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172,189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376, 393, 410,427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597, 614, 631, 648,665, 682, 699, 716, 733, 750, 767, 784, 801, 818, 835, 852, 869, 886,903, 920, 937, 954, 971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107,1124, 1141, 1158, 1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311,1328, 1345, 1362, 1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515,1532, 1549, and 1566 and/or a recombinant, purified, and/or isolatedpolypeptide selected from the group consisting of a polypeptide havingat least 90% sequence identity to at least one of the sequences of SEQID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222,239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460,477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698,715, 732, 749, 766, 783, 800, 817, 834, 851, 868, 885, 902, 819, 936,953, 970, 987, 1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123, 1140,1157, 1174, 1191, 1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327, 1344,1361, 1378, 1395, 1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531, 1548,and 1565 to a cell.

In embodiments of the invention any one of the isolated and/or purifiedpolypeptides according to the invention may be enzymatically orfunctionally active at temperatures at or above about 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and/or 95 degrees Celsius and/ormay be enzymatically or functionally active at a pH at, below, and/orabove 8, 7, 6, 5, 4, 3, 2, 1, and/or 0. In further embodiments of theinvention, glycosylation, pegylation, and/or other post-translationalmodification may be required for the isolated and/or purifiedpolypeptides according to the invention to be enzymatically orfunctionally active at a pH at or below 8, 7, 6, 5, 4, 3, 2, 1, and/or 0or at temperatures at or above about 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, and/or 95 degrees Celsius.

The invention is described in additional detail in the followingillustrative examples. Although the examples may represent only selectedembodiments of the invention, it should be understood that the followingexamples are illustrative and not limiting.

EXAMPLES Example 1: Modulating or Altering Metabolism Using Nucleotideand Amino Acid Sequences from Alicyclobacillus acidocaldarius

Provided in SEQ ID NOS:2, 19, 36, 53, 70, 87, 104, 121, 138, 155, 172,189, 206, 223, 240, 257, 274, 291, 308, 325, 342, 359, 376, 393, 410,427, 444, 461, 478, 495, 512, 529, 546, 563, 580, 597, 614, 631, 648,665, 682, 699, 716, 733, 750, 767, 784, 801, 818, 835, 852, 869, 886,903, 920, 937, 954, 971, 988, 1005, 1022, 1039, 1056, 1073, 1090, 1107,1124, 1141, 1158, 1175, 1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311,1328, 1345, 1362, 1379, 1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515,1532, 1549, and 1566 are a nucleotide sequence isolated fromAlicyclobacillus acidocaldarius and coding for the polypeptides of SEQID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222,239, 256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460,477, 494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698,715, 732, 749, 766, 783, 800, 817, 834, 851, 868, 885, 902, 819, 936,953, 970, 987, 1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123, 1140,1157, 1174, 1191, 1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327, 1344,1361, 1378, 1395, 1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531, 1548,and 1565, respectively. The nucleotide sequences of SEQ ID NOS:2, 19,36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257, 274,291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495, 512,529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733, 750,767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954, 971, 988,1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158, 1175, 1192,1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362, 1379, 1396,1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and 1566 areplaced into expression vectors using techniques standard in the art. Thevectors are then provided to cells such as bacteria cells or eukaryoticcells such as SD cells or CHO cells. In conjunction with the normalmachinery in present in the cells, the vectors comprising SEQ ID NOS:2,19, 36, 53, 70, 87, 104, 121, 138, 155, 172, 189, 206, 223, 240, 257,274, 291, 308, 325, 342, 359, 376, 393, 410, 427, 444, 461, 478, 495,512, 529, 546, 563, 580, 597, 614, 631, 648, 665, 682, 699, 716, 733,750, 767, 784, 801, 818, 835, 852, 869, 886, 903, 920, 937, 954, 971,988, 1005, 1022, 1039, 1056, 1073, 1090, 1107, 1124, 1141, 1158, 1175,1192, 1209, 1226, 1243, 1260, 1277, 1294, 1311, 1328, 1345, 1362, 1379,1396, 1413, 1430, 1447, 1464, 1481, 1498, 1515, 1532, 1549, and 1566produce the polypeptides of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120,137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307, 324, 341, 358,375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545, 562, 579, 596,613, 630, 647, 664, 681, 698, 715, 732, 749, 766, 783, 800, 817, 834,851, 868, 885, 902, 819, 936, 953, 970, 987, 1004, 1021, 1038, 1055,1072, 1089, 1106, 1123, 1140, 1157, 1174, 1191, 1208, 1225, 1242, 1259,1276, 1293, 1310, 1327, 1344, 1361, 1378, 1395, 1412, 1429, 1446, 1463,1480, 1497, 1514, 1531, 1548, and 1565. The polypeptides of SEQ IDNOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239,256, 273, 290, 307, 324, 341, 358, 375, 392, 409, 426, 443, 460, 477,494, 511, 528, 545, 562, 579, 596, 613, 630, 647, 664, 681, 698, 715,732, 749, 766, 783, 800, 817, 834, 851, 868, 885, 902, 819, 936, 953,970, 987, 1004, 1021, 1038, 1055, 1072, 1089, 1106, 1123, 1140, 1157,1174, 1191, 1208, 1225, 1242, 1259, 1276, 1293, 1310, 1327, 1344, 1361,1378, 1395, 1412, 1429, 1446, 1463, 1480, 1497, 1514, 1531, 1548, and1565 are then isolated and/or purified. The isolated and/or purifiedpolypeptides of SEQ ID NOS:1, 18, 35, 52, 69, 86, 103, 120, 137, 154,171, 188, 205, 222, 239, 256, 273, 290, 307, 324, 341, 358, 375, 392,409, 426, 443, 460, 477, 494, 511, 528, 545, 562, 579, 596, 613, 630,647, 664, 681, 698, 715, 732, 749, 766, 783, 800, 817, 834, 851, 868,885, 902, 819, 936, 953, 970, 987, 1004, 1021, 1038, 1055, 1072, 1089,1106, 1123, 1140, 1157, 1174, 1191, 1208, 1225, 1242, 1259, 1276, 1293,1310, 1327, 1344, 1361, 1378, 1395, 1412, 1429, 1446, 1463, 1480, 1497,1514, 1531, 1548, and 1565 are then each demonstrated to have one ormore of the activities provided in Table 1.

The isolated and/or purified polypeptides of SEQ ID NOS:1, 18, 35, 52,69, 86, 103, 120, 137, 154, 171, 188, 205, 222, 239, 256, 273, 290, 307,324, 341, 358, 375, 392, 409, 426, 443, 460, 477, 494, 511, 528, 545,562, 579, 596, 613, 630, 647, 664, 681, 698, 715, 732, 749, 766, 783,800, 817, 834, 851, 868, 885, 902, 819, 936, 953, 970, 987, 1004, 1021,1038, 1055, 1072, 1089, 1106, 1123, 1140, 1157, 1174, 1191, 1208, 1225,1242, 1259, 1276, 1293, 1310, 1327, 1344, 1361, 1378, 1395, 1412, 1429,1446, 1463, 1480, 1497, 1514, 1531, 1548, and 1565 are demonstrated tohave activity as at least one of a (S)-2-hydroxy-acid oxidase,[acyl-carrier-protein] S-malonyltransferase, 1,3-propanediolDehydrogenase, 2-isopropylmalate Synthase, 3-hydroxybutyryl-CoAdehydratase, 3-isopropylmalate Dehydratase, 3-isopropylmalateDehydrogenase, 3-oxoacid CoA-transferase, 8-amino-7-oxononanoateSynthase, Acetaldehyde dehydrogenase (acetylating), Acetate-CoA ligase,Acetolactate synthase, Acetyl-CoA C-acetyltransferase, Aconitatehydratase, Alcohol dehydrogenase, Alcohol dehydrogenase (NADP+),Aldehyde dehydrogenase, Aldehyde dehydrogenase (NAD+), ATPphosphoribosyltransferase, ATP synthase alpha chain, ATP synthase Bchain, ATP synthase beta chain, ATP synthase C chain, ATP synthaseepsilon chain, ATP synthase gamma chain, Biotin synthase,Branched-chain-amino-acid transaminase, Butyryl-CoA dehydrogenase,Citrate (Si)-synthase, Dethiobiotin synthase, Diaminopimelatedecarboxylase, Diaminopimelate epimerase, Dihydrodipicolinate reductase,Dihydrodipicolinate synthase, Dihydrolipoyl dehydrogenase,Dihydroxy-acid dehydratase, Enoyl-CoA hydratase, FdhD protein (fdsC),Formate dehydrogenase, Glycerate kinase, Glycinehydroxymethyltransferase, Isocitrate lyase, Lactaldehyde reductase,Lactate 2-monooxygenase, L-lactate dehydrogenase, Malate dehydrogenase,Malate dehydrogenase (acceptor), Malate dehydrogenase(oxaloacetate-decarboxylating), Malate synthase, Malonate-semialdehydedehydrogenase (acetylating), Methylm al onate-semi al dehydedehydrogenase (acylating), N-acetyldiaminopimelate deacetylase,Oxoglutarate dehydrogenase (succinyl-transferring), Phosphoenolpyruvatecarboxylase, Phosphoglycerate dehydrogenase, Phosphoribosylanthranilateisomerase, Pyruvate dehydrogenase (acetyl-transferring), Pyruvate,phosphate dikinase, Succinate dehydrogenase cytochrome b558 subunit,Succinate dehydrogenase flavoprotein subunit, Succinate dehydrogenaseiron-sulfur protein, and Succinate-CoA ligase (ADP-forming).

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and that fallwithin the limits of the appended claims and their legal equivalents.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein.

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and that fallwithin the limits of the appended claims and their legal equivalents.

BIBLIOGRAPHIC REFERENCES

-   Barany, F., 1991, PNAS USA, 88:189-193.-   Buckholz, R. G., 1993, Yeast systems for the expression of    heterologous gene products. Curr. Op. Biotechnology 4:538-542.-   Burg, J. L. et al., 1996, Mol. and Cell. Probes, 10:257-271.-   Chu, B. C. F. et al., 1986, NAR, 14:5591-5603.-   Duck, P. et al., 1990, Biotechniques, 9:142-147.-   Edwards, C. P., and Aruffo, A., 1993, Current applications of COS    cell based transient expression systems, Curr. Op. Biotechnology    4:558-563.-   Guateli, J. C. et al., 1990, PNAS USA, 87:1874-1878.-   Houben-Weyl, 1974, in Methoden der Organischen Chemie, E. Wunsch    Ed., Volume 15-I and 15-II, Thieme, Stuttgart.-   Innis, M. A. et al., 1990, in PCR Protocols, A guide to Methods and    Applications, San Diego, Academic Press.-   Kievitis, T. et al., 1991, J. Virol. Methods, 35:273-286.-   Köhler, G. et al., 1975, Nature, 256(5517):495-497.-   Kwoh, D. Y. et al., 1989, PNAS USA, 86:1173-1177.-   Luckow, V. A., 1993, Baculovirus systems for the expression of human    gene products. Curr. Op. Biotechnology 4:564-572.-   Matthews, J. A. et al., 1988, Anal. Biochem., 169:1-25.-   Merrifield, R. D., 1966, J. Am. Chem. Soc., 88(21):5051-5052.-   Miele, E. A. et al., 1983, J. Mol. Biol., 171:281-295.-   Olins, P. O., and Lee, S. C., 1993, Recent advances in heterologous    gene expression in E. coli. Curr. Op. Biotechnology 4:520-525.-   Rolfs, A. et al., 1991, In PCR Topics, Usage of Polymerase Chain    reaction in Genetic and Infectious Disease, Berlin: Springer-Verlag.-   Sambrook, J. et al., 1989, In Molecular Cloning: A Laboratory    Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory    Press.-   Sanchez-Pescador, R., 1988, J. Clin. Microbiol., 26(10):1934-1938.-   Segev D., 1992, in “Non-radioactive Labeling and Detection of    Biomolecules,” Kessler C. Springer-Verlag, Berlin, New-York:    197-205.-   Urdea, M. S., 1988, Nucleic Acids Research, 11:4937-4957.-   Walker, G. T. et al., 1992, NAR 20:1691-1696.-   Walker, G. T. et al., 1992, PNAS USA, 89:392-396.-   White, B. A. et al., 1997, Methods in Molecular Biology, 67, Humana    Press, Totowa, N.J.

1.-8. (canceled)
 9. An expression vector comprising an isolatedpolynucleotide encoding a polypeptide having at least 90% sequenceidentity to SEQ ID No. 427 and a nucleotide sequence heterologous to thepolynucleotide.
 10. The expression vector of claim 9, wherein theexpression vector comprises an isolated polynucleotide having at least95% identity to SEQ ID No.
 426. 11. The expression vector of claim 9,wherein the encoded polypeptide has Butyryl-CoA dehydrogenase enzymaticactivity.
 12. A method of modulating or altering metabolism in a cell,the method comprising: providing the expression vector of claim 11 tothe cell and expressing the encoded polypeptide in the cell.
 13. Themethod according to claim 12, further comprising glycosylating, orotherwise post-translationally modifying the encoded peptide in thecell.
 14. An expression vector comprising an isolated polynucleotideencoding a polypeptide having at least 95% sequence identity to SEQ IDNo. 427 and a nucleotide sequence heterologous to the polynucleotide.15. The expression vector of claim 14, wherein the encoded polypeptidehas Butyryl-CoA dehydrogenase enzymatic activity.
 16. The expressionvector of claim 14, wherein the expression vector comprises an isolatedpolynucleotide having at least 95% identity to SEQ ID No. 426.